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EP4104187A1 - Procédé de prédiction de réponse à une thérapie de récepteur antigénique chimérique - Google Patents

Procédé de prédiction de réponse à une thérapie de récepteur antigénique chimérique

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Publication number
EP4104187A1
EP4104187A1 EP21714998.8A EP21714998A EP4104187A1 EP 4104187 A1 EP4104187 A1 EP 4104187A1 EP 21714998 A EP21714998 A EP 21714998A EP 4104187 A1 EP4104187 A1 EP 4104187A1
Authority
EP
European Patent Office
Prior art keywords
car
lesion
therapy
cell
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21714998.8A
Other languages
German (de)
English (en)
Inventor
Jayaram K. Udupa
Drew A. TORIGIAN
Stephen Schuster
Yubing TONG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
University of Pennsylvania Penn
Original Assignee
Novartis AG
University of Pennsylvania Penn
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novartis AG, University of Pennsylvania Penn filed Critical Novartis AG
Publication of EP4104187A1 publication Critical patent/EP4104187A1/fr
Pending legal-status Critical Current

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/50—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for simulation or modelling of medical disorders
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/20—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for computer-aided diagnosis, e.g. based on medical expert systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T7/00—Image analysis
    • G06T7/0002—Inspection of images, e.g. flaw detection
    • G06T7/0012—Biomedical image inspection
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V10/00—Arrangements for image or video recognition or understanding
    • G06V10/70—Arrangements for image or video recognition or understanding using pattern recognition or machine learning
    • G06V10/764—Arrangements for image or video recognition or understanding using pattern recognition or machine learning using classification, e.g. of video objects
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V10/00—Arrangements for image or video recognition or understanding
    • G06V10/70—Arrangements for image or video recognition or understanding using pattern recognition or machine learning
    • G06V10/82—Arrangements for image or video recognition or understanding using pattern recognition or machine learning using neural networks
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H30/00—ICT specially adapted for the handling or processing of medical images
    • G16H30/40—ICT specially adapted for the handling or processing of medical images for processing medical images, e.g. editing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/20—Special algorithmic details
    • G06T2207/20084—Artificial neural networks [ANN]
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30096—Tumor; Lesion
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06V—IMAGE OR VIDEO RECOGNITION OR UNDERSTANDING
    • G06V2201/00—Indexing scheme relating to image or video recognition or understanding
    • G06V2201/03—Recognition of patterns in medical or anatomical images
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the present invention relates generally to methods of predicting response to Chimeric Antigen Receptor (CAR) therapy.
  • CAR Chimeric Antigen Receptor
  • the present disclosure pertains, at least in part, to a method, e.g., a computer- implemented method, for determining, e.g., predicting, a lesion-level treatment response to a chimeric antigen receptor (CAR) therapy, e.g., a therapy comprising an immune effector cell or population of immune effector cells that expresses a CAR that binds to CD 19 (also referred to herein as a “CAR 19 therapy” or a “CD 19 CAR therapy”).
  • CAR chimeric antigen receptor
  • the method further comprises a rule- based reasoning methodology for patient-level response prediction.
  • a system and a non-transitory computer-readable medium for determining a lesion-level response.
  • the disclosure provides a method for treating a subject having, or at risk of having a lymphoma (e.g., a lymphoma described herein, e.g., DLBCL or FL), by administering a CAR19 therapy to the subject responsive to a determination, e.g., prediction, of a lesion-level treatment response to the CAR 19 therapy.
  • a lymphoma e.g., a lymphoma described herein, e.g., DLBCL or FL
  • a determination e.g., prediction
  • a lesion-level treatment response to the CAR 19 therapy e.g., a lesion-level treatment response to the CAR 19 therapy.
  • the disclosure provides a computer-implemented method for determining, e.g., predicting, a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”), said method comprising: acquiring, e.g., receiving, an image of a lesion of a subject, e.g., a subject having or at risk of having a lymphoma (“acquired image”); and processing the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a neural network e.g., a neural network
  • a system for determining, e.g., predicting, a lesion- level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD 19 (a “CAR19 therapy”)
  • said system comprising: a processor; and a storage device storing instructions that, when executed by the processor, cause the processor to: acquire, e.g., receive, an image of a lesion of a subject, e.g., a subject having, or at risk of having, a lymphoma (“acquired image”); and process the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a neural network e.g., a neural network
  • the disclosure provides a non-transitory computer-readable medium for determining, e.g., predicting, a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”), said medium comprising instructions that, when executed by a processor, cause the processor to: acquire, e.g., receive, an image of a lesion of the subject, e.g., a subject having, or at risk of having, a lymphoma (“acquired image”); and process the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a neural network e.g., a neural network
  • a method for treating a subject having, or at risk of having, lymphoma comprising: responsive to a determination, e.g., prediction, of a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”), administering the CAR 19 therapy to the subject, thereby treating the subject, wherein said determination, e.g., prediction, comprises: acquiring, e.g., receiving, an image of a lesion of the subject (“acquired image”); and processing the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a determination e.g., prediction
  • the disclosure provides a method for evaluating, or predicting the responsiveness of, a subject having or at risk of having a lymphoma to a CAR19 therapy, said method comprising: determining, e.g., predicting, of a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”), with a neural network, wherein said determining comprises: acquiring, e.g., receiving, an image of a lesion of the subject (“acquired image”); and processing the image with the neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy; and thereby evaluating the subject, or predicting the responsiveness of the subject, to the CAR 19 therapy.
  • a neural network comprises: acquiring, e.g., receiving, an image of a lesion of
  • the CAR19 therapy is a therapy comprising immune effector cells expressing an anti-CD 19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain.
  • the lymphoma e.g., relapsed and/or refractory lymphoma
  • the lymphoma is chosen from diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL).
  • the lymphoma is DLBCL, e.g., relapsed and/or refractory DLBCL.
  • the lymphoma is FL, e.g., relapsed and/or refractory FL.
  • the lesion-level treatment response is indicative of responsiveness to the CAR 19 therapy.
  • the lesion-level treatment response is evaluated using one, two, three or more (all) of the following parameters: 1) a change in lesion size; 2) a change in metabolic activity; 3) a change in lesion morphology; or 4) a change in lesion density.
  • the lesion-level treatment response is evaluated using a change in lesion size.
  • the lesion-level treatment response is evaluated using a change in metabolic activity.
  • the lesion- level treatment response is evaluated using a change in lesion morphology.
  • the lesion-level treatment response is evaluated using a change in lesion density.
  • a decrease in one or more of 1-4 is indicative of a positive response to the CAR 19 therapy.
  • an increase or lack of a detectable change in one or more of 1-4 is indicative of a negative response to the CAR 19 therapy.
  • the prediction of the lesion-level treatment response is followed by a rule-based reasoning method, to thereby determine a patient-level response prediction.
  • the patient-level response prediction comprises an All rule and/or a Majority Rule.
  • the patient-level response prediction comprises an All rule.
  • the patient-level response prediction comprises a Majority rule.
  • a computer-implemented method for determining, e.g., predicting, a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD 19 comprising: acquiring, e.g., receiving, an image of a lesion of a subject, e.g., a subject having or at risk of having a lymphoma (“acquired image”); and processing the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a system for determining, e.g., predicting, a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”)
  • said system comprising: a processor; and a storage device storing instructions that, when executed by the processor, cause the processor to: acquire, e.g., receive, an image of a lesion of a subject, e.g., a subject having, or at risk of having, a lymphoma (“acquired image”); and process the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a neural network e.g., a neural network
  • a non-transitory computer-readable medium for determining, e.g., predicting, a lesion- level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD 19 (a “CAR19 therapy”), said medium comprising instructions that, when executed by a processor, cause the processor to: acquire, e.g., receive, an image of a lesion of the subject, e.g., a subject having, or at risk of having, a lymphoma (“acquired image”); and process the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a neural network e.g., a neural network
  • a method for treating a subject having, or at risk of having, lymphoma comprising: responsive to a determination, e.g., prediction, of a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”), administering the CAR 19 therapy to the subject, thereby treating the subject, wherein said determination, e.g., prediction, comprises: acquiring, e.g., receiving, an image of a lesion of the subject (“acquired image”); and processing the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • a determination e.g., prediction
  • a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (
  • a method for evaluating, or predicting the responsiveness of, a subject having or at risk of having a lymphoma to a CAR19 therapy comprising: determining, e.g., predicting, of a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”), with a neural network, wherein said determining comprises: acquiring, e.g., receiving, an image of a lesion of the subject (“acquired image”); and processing the image with the neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy; and thereby evaluating the subject, or predicting the responsiveness of the subject, to the CAR 19 therapy.
  • determining e.g., predicting, of a lesion-level treatment response to a therapy comprising an immune effector cell or
  • the CAR19 therapy is a therapy comprising immune effector cells expressing an anti-CD 19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain.
  • lymphoma is chosen from diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma.
  • DLBCL diffuse large B-cell lymphoma
  • follicular lymphoma follicular lymphoma
  • the acquired image is a post-treatment image, e.g., after a CAR19 therapy and/or a different therapy (e.g., chemotherapy or radiotherapy).
  • a different therapy e.g., chemotherapy or radiotherapy.
  • the acquired image comprises a plurality of images, e.g., at least two images of different views of the same lesion in the subject.
  • CT computed tomography
  • dCT diagnostic CT
  • /CT low dose CT
  • MRI magnetic resonance imaging
  • SPECT single-photon emission computerized tomography
  • the acquired image is one of a single volume of interest (VOI)-restricted image slice passing through a mid-portion of a lesion, three contiguous VOTrestricted image slices passing through a mid-portion of a lesion, a single whole image slice passing through a mid portion of a lesion, three contiguous whole image slices passing through a mid-portion of a lesion, or a single VOT slice and a single whole slice passing through a mid portion of a lesion and combined into two channels of one input sample, or a combination thereof.
  • VI volume of interest
  • the acquired image comprises an image of at least one, two, three, four, five, ten, twenty, fifty, one hundred, five hundred or one thousand lesions in the subject.
  • the lesion-level treatment response is evaluated using one or more of the following parameters: 1) a change in lesion size; 2) a change in lesion metabolic activity; 3) a change in lesion morphology; 4) a change in lesion intensity (e.g., lesion attenuation (on CT), lesion signal intensity (on MRI, whether T1 -weighted, T2-weighted, or diffusion-weighted), or lesion contrast enhancement (on CT or MRI)); 5) a change in lesion morphology (e.g., a lesion size, a lesion volume, or a lesion shape); 6) a change in lesion radiotracer uptake (e.g., FDG uptake on PET imaging or DOTATE uptake on PET imaging); 7) a change in lesion texture (e.g., on CT, MRI, PET, or SPECT); and/or 8) a change in non-lesion tissue properties (e.
  • lesion intensity e.g., lesion attenuation (
  • a responder in the All rule of the patient-level response prediction is a subject in whom all evaluated lesions have responded, or are predicted to respond to the CAR 19 therapy.
  • a non-responder in the All rule of the patient-level response prediction is a subject in whom at least one evaluated lesion has not responded, or is predicted not to respond to the CAR 19 therapy.
  • a responder in the Majority rule of the patient-level response prediction is a subject in whom a majority of the evaluated lesions (e.g., based on a % threshold, e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have responded, or are predicted to respond to the CAR19 therapy.
  • a non responder in the Majority rule of the patient-level response prediction is a subject in whom a majority of the evaluated lesions have not responded (e.g., based on a % threshold, e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have not responded, or are predicted not to respond to the CAR 19 therapy.
  • a % threshold e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have not responded, or are predicted not to respond to the CAR 19 therapy.
  • the responder according to the patient-level response prediction is a subject who has, or has shown a clinical response, e.g., a detectable clinical response, e.g., a complete response or a partial response.
  • a responder according to the patient- level response prediction corresponds to a subject having a low score in a clinical stratification tool, e.g., International Prognostic Index (IPI) for DLBCL or follicular lymphoma international prognostic index (FLIPI) for follicular lymphoma.
  • IPI International Prognostic Index
  • FLIPI international prognostic index
  • a non responder according to the patient-level response prediction corresponds to a subject having a high score in a clinical stratification tool, e.g., International Prognostic Index (IPI) for DLBCL or follicular lymphoma international prognostic index (FLIPI) for follicular lymphoma.
  • IPI International Prognostic Index
  • FLIPI international prognostic index
  • the plurality of layers comprises at least two of: an input layer, a convolutional layer, a fully connected layer, a pooling layer, a classification layer, and an output layer.
  • CNN convolutional neural network
  • the pre-trained convolutional neural network is one of VGGNet, AlexNet, ResNet, SqueezeNet, or GoogLeNet.
  • the VGGNet is VGG-16 or VGG-19.
  • the neural network is trained according to: loading a pre-trained neural network; modifying at least one layer of the pre-trained neural network, wherein the modifying includes replacing the at least one layer with at least one of a fully connected layer, a softmax layer, or a binary classification output layer, thereby creating a modified neural network; loading a set of labeled pre-treatment digital images; and re-training the modified neural network using the set of labeled pre-treatment images.
  • the neural network is trained according to: loading the pre-trained neural network comprising an input layer, a first convolutional layer with ReLU (rectified linear unit), a first max pooling layer, a second convolutional layer with ReLU, a second max pooling layer, a third convolutional layer with ReLU, a fourth convolutional layer with ReLU, a third max pooling layer, a first fully connected layer with ReLU and dropout, a second fully connected layer with ReLU and dropout, a third fully connected layer, a softmax layer, and an output layer; and replacing the third fully connected layer, the softmax layer, and the output layer with, respectively, a new fully connected layer, a new softmax layer, and a binary classification output layer, thereby forming the modified neural network.
  • ReLU rectified linear unit
  • replacing the at least one layer comprises: replacing three layers of the pre-trained neural network with a fully connected layer, a softmax layer, and a binary classification output layer.
  • parameters of the modified neural network are optimized using Stochastic Gradient Descent (SGD), Stochastic Gradient Descent with momentum, Simulated Annealing (SA), or Conjugate Gradient (CG).
  • CT computed tomography
  • dCT diagnostic CT
  • /CT low dose CT
  • MRI magnetic resonance imaging
  • SPECT single-photon emission computerized tomography
  • PET/CT PET/CT
  • PET/MRI PET/MRI
  • SPECT/CT SPECT/CT
  • CT/PET single-photon emission computerized tomography
  • the set of labeled pre-treatment digital images is pre-processed by performing mean subtracting and down sampling using bilinear interpolation on each image of the set of labeled pre-treatment digital images.
  • testing set is independent of the validation set and the training set.
  • the modified neural network receives, at an input of the modified neural network, an input of: a single volume of interest (VOI)-restricted image slice passing through a mid-portion of a lesion, three contiguous VOTrestricted image slices passing through a mid-portion of a lesion, a single whole image slice passing through a mid portion of a lesion, three contiguous whole image slices passing through a mid-portion of a lesion, or a single VOTslice and a single whole slice combined into two channels of one input sample, or a combination thereof.
  • VI volume of interest
  • modified neural network is re-trained anew for each of a single volume of interest (VOI)-restricted image slice passing through a mid-portion of a lesion, three contiguous VOTrestricted image slices passing through a mid-portion of a lesion, a single whole image slice passing through a mid portion of a lesion, three contiguous whole image slices passing through a mid-portion of a lesion and a single VOTslice and a single whole slice combined into two channels of one input sample.
  • VI volume of interest
  • the incremental transfer learning comprises: loading an additional set of labeled pre-treatment images; and performing at least a second re-training by re-training the modified neural network using the additional set of labeled pre-treatment images.
  • CT computed tomography
  • dCT diagnostic CT
  • /CT low dose CT
  • MRI magnetic resonance imaging
  • SPECT single-photon emission computerized tomography
  • CT computed tomography
  • dCT diagnostic CT
  • /CT low dose CT
  • MRI magnetic resonance imaging
  • SPECT single-photon emission computerized tomography
  • the modified neural network receives, at an input of the modified neural network, an input of: a single volume of interest (V derestricted image slice passing through a mid-portion of a lesion, three contiguous VOTrestricted image slices passing through a mid-portion of a lesion, a single whole image slice passing through a mid portion of a lesion, three contiguous whole image slices passing through a mid-portion of a lesion, or a single VOT slice and a single whole slice combined into two channels of one input sample, or a combination thereof.
  • a single volume of interest V derestricted image slice passing through a mid-portion of a lesion, three contiguous VOTrestricted image slices passing through a mid-portion of a lesion, a single whole image slice passing through a mid portion of a lesion, three contiguous whole image slices passing through a mid-portion of a lesion, or a single VOT slice and a single whole slice combined into two channels of one input sample, or
  • modified neural network is re- trained anew for each of a single whole image slice passing through mid-portions of lesions and three contiguous whole image slices passing through mid-portions of lesions.
  • a responder is a subject who has a clinical response, e.g., a detectable clinical response, e.g., a complete response or a partial response.
  • a non-responder according to the patient-level response prediction corresponds to a subject having a high score in a clinical stratification tool, e.g., International Prognostic Index (IPI) for DLBCL or follicular lymphoma international prognostic index (FLIPI) for follicular lymphoma.
  • IPI International Prognostic Index
  • FLIPI international prognostic index
  • the method further comprises comparing the results of the patient-level prediction with a clinical stratification tool, e.g., IPI or FLIPI.
  • a clinical stratification tool e.g., IPI or FLIPI.
  • the CAR comprises an antibody molecule which includes an anti-CD 19 binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain
  • said anti-CD 19 binding domain comprises one or more of light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of any anti-CD19 light chain binding domain amino acid sequence listed in Table 3B, and one or more of heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of any anti-CD19 heavy chain binding domain amino acid sequence listed in Table 3A.
  • LC CDR1 light chain complementary determining region 1
  • HC CDR2 light chain complementary determining region 2
  • HC CDR3 heavy chain complementary determining region 3
  • the CAR is a CD19 CAR, e.g., a CAR comprising an scFv amino acid sequence listed in Table 3, e.g., amino acid sequence of SEQ ID NO: 39-51 or a CAR comprising the amino acid sequence of SEQ ID NO: 77-89.
  • anti-CD19 binding domain comprises the sequence of SEQ ID NO: 40, or SEQ ID NO:51.
  • the CAR comprises a polypeptide having the sequence of SEQ ID NO:78, or SEQ ID NO: 89.
  • the CAR 19 therapy is a therapy comprising one or more of CTL-019, CTL-119, and CAR 2A.
  • CAR is a CD19 CAR, e.g., a CAR comprising an scFv amino acid sequence listed in Table 3, e.g., amino acid sequence of SEQ ID NO: 144 or SEQ ID NO: 147, or a CAR comprising the amino acid sequence of SEQ ID NO: 143 or SEQ ID NO: 146.
  • CD19 CAR e.g., a CAR comprising an scFv amino acid sequence listed in Table 3, e.g., amino acid sequence of SEQ ID NO: 144 or SEQ ID NO: 147, or a CAR comprising the amino acid sequence of SEQ ID NO: 143 or SEQ ID NO: 146.
  • anti-CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 144 or SEQ ID NO: 147.
  • FIG. 1 is a schematic showing the subject inclusion and exclusion criteria for the study described in Example 1.
  • FIG. 2 shows the deep learning-based architecture for lesion-level treatment response prediction.
  • FIG.3 shows the strategy of transfer learning and incremental learning on lesion-level prediction.
  • FIG. 4 shows receiver operator characteristic (ROC) curves for diagnostic performance of lesion-level treatment response prediction using transfer learning for 4 scenarios (1 VOI, 1 slice, 3 VOIs, 3 slices) on 3 imaging modalities (diagnostic computed tomography (dCT), low- dose computed tomography (1CT), and positron emission tomography (PET) images).
  • dCT diagnostic computed tomography
  • CT low- dose computed tomography
  • PET positron emission tomography
  • FIG. 5 shows receiver operator characteristic (ROC) curves for diagnostic performance of lesion-level treatment response prediction using transfer learning for 1 VOI plus 1 slice and 1 slice scenarios on diagnostic computed tomography (dCT), low-dose computed tomography (1CT), and positron emission tomography (PET) images.
  • dCT diagnostic computed tomography
  • 1CT low-dose computed tomography
  • PET positron emission tomography
  • FIG. 6 shows receiver operator characteristic (ROC) curves for diagnostic performance of lesion-level treatment response prediction using incremental learning vs. transfer learning for 1 slice and 3 slice scenarios on diagnostic computed tomography (dCT), low-dose computed tomography (1CT), and positron emission tomography (PET) images.
  • dCT diagnostic computed tomography
  • 1CT low-dose computed tomography
  • PET positron emission tomography
  • FIG. 8 is a block diagram of a distributed computer system 800 including a computer system 802.
  • FIG. 9 is flowchart depicting the transfer learning method 900.
  • FIG. 10 is flowchart depicting the incremental learning method 1000. DETAILED DESCRIPTION
  • Described herein, inter alia, is a method for determining, e.g., predicting, a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD 19 (a “CAR19 therapy”), based, e.g., on deep-learning (DL) image analysis.
  • the method further comprises a rule-based reasoning methodology for patient-level response prediction.
  • the medical image-based approach disclosed herein to determine personalized response prediction to CAR T-cell therapies has several unique advantages over conventional methods including, but not limited to: (1) the use of pre-existing diagnostic imaging data sets previously acquired for clinical purposes; (2) the lack of invasiveness; (3) the extraction of regional phenotypic information from disease and extra-disease sites throughout the body that may be heterogeneous; and (4) production-mode efficiency.
  • the disclosure provides a method of treating a subject having, or at risk of having a lymphoma (e.g. DLBCL or FL), wherein responsive to a determination, e.g., prediction, of a lesion-level treatment response to a therapy comprising an immune effector cell or population of immune effector cells that expresses a Chimeric Antigen Receptor (CAR) that binds to CD19 (a “CAR19 therapy”), administering the CAR 19 therapy to the subject, thereby treating the subject.
  • a lymphoma e.g. DLBCL or FL
  • a determination e.g., prediction
  • CAR Chimeric Antigen Receptor
  • Also disclosed herein is a method of evaluating a subject having, or at risk of having a lymphoma, comprising determining, e.g., predicting, of a lesion-level treatment response to a therapy comprising a Chimeric Antigen Receptor 19 (CAR19) immune effector cell (“CAR19 therapy”), with a neural network.
  • the determination comprises: acquiring, e.g., receiving, an image of a lesion of the subject (“acquired image”); and/or processing the image with the neural network (“processed image”).
  • the neural network outputs a classification result indicating the lesion-level treatment response to the CAR19 therapy.
  • lesion-level treatment response refers to a prediction and/or evaluation of responsiveness of a lesion to a therapy.
  • the lesion is in a subject or in a sample from the subject.
  • a lesion-level treatment response comprises an output from a neural network, e.g., as described herein.
  • a lesion-level treatment response is indicative of responsiveness to a therapy, e.g., a CAR19 therapy.
  • the lesion-level treatment response is evaluated using one, two, three or all of the following parameters: 1) a change in lesion size; 2) a change in metabolic activity; 3) a change in lesion morphology; or 4) a change in lesion density.
  • classification result refers to an output from a neural network.
  • a classification result is a binary classification result.
  • patient-level response refers to a prediction of responsiveness of a subject (e.g., patient) to a therapy.
  • the therapy is a CAR19 therapy.
  • the patient-level response comprises a rule-based reasoning method which, e.g., is based on a lesion-level treatment response.
  • the patient-level response comprises an All rule or a Majority rule.
  • a patient- level response prediction classifies a subject as a responder or a non-responder.
  • a responder is a subject who has, or has shown a clinical response, e.g., a detectable clinical response, e.g., a complete response or a partial response.
  • a non-responder is a subject who does not have, or has not shown a clinical response, e.g., does not have a detectable clinical response, e.g., has progressive disease.
  • a pre-determined threshold for a responder according to the All rule is a subject in whom all evaluated lesions have responded, or are predicted to respond to the therapy, e.g., CAR19 therapy.
  • a pre-determined threshold for a responder according to the Majority rule is a subject in whom a majority of the evaluated lesions (e.g., based on a % threshold, e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have responded, or are predicted to respond to the therapy, e.g., CAR19 therapy.
  • a majority of the evaluated lesions e.g., based on a % threshold, e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have responded, or are predicted to respond to the therapy, e.g., CAR19 therapy.
  • a pre-determined threshold for a responder according to a clinical response criterion is a subject who has, or has shown a clinical response, e.g., a detectable clinical response, e.g., a complete response or a partial response.
  • a pre-determined threshold for a responder according to the International Prognostic Index (IPI) for DLBCL is a subject having a high score, e.g., a score of >2.
  • a pre-determined threshold for a responder according to the follicular lymphoma international prognostic index (FLIPI) is a subject having a high score, e.g., a score of > 2.
  • non-responder refers to a subject who meets a pre determined threshold according to the All rule, the Majority rule, a prognostic index (e.g., a patient stratification tool) and/or a clinical response criterion.
  • a pre determined threshold for a non-responder according to the All rule is subject in whom at least one evaluated lesion has not responded, or is predicted not to respond to the therapy, e.g.,
  • a pre-determined threshold for a non-responder according to the Majority rule a subject in whom a majority of the evaluated lesions have not responded (e.g., based on a % threshold, e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have not responded, or are predicted not to respond to the therapy, e.g., CAR19 therapy.
  • a pre-determined threshold for a non-responder according to clinical response criteria is a subject who does not have, or has not shown a clinical response, e.g., does not have a detectable clinical response, e.g., has progressive disease.
  • a pre determined threshold for a non-responder according to the International Prognostic Index (IPI) for DLBCL is a subject having a low score, e.g., a score of ⁇ 2.
  • a pre determined threshold for a non-responder according to the follicular lymphoma international prognostic index (FLIPI) is a subject having a low score, e.g., a score of ⁇ 2.
  • CAR Chimeric Antigen Receptor
  • a CAR refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation.
  • a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below.
  • the set of polypeptides are in the same polypeptide chain (e.g., comprise a chimeric fusion protein). In some embodiments, the set of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In one embodiment, the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex.
  • the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In one embodiment, the cytoplasmic signaling domain further comprises one or more functional signaling domains of at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, ICOS, and/or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain of a stimulatory molecule.
  • 4-1BB i.e., CD137
  • CD27 CD27
  • ICOS ICOS
  • CD28 CD28
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain of a co-stimulatory molecule and a functional signaling domain of a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains of one or more co- stimulatory molecule(s) and a functional signaling domain of a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains of one or more co-stimulatory molecule(s) and a functional signaling domain of a stimulatory molecule.
  • the CAR comprises an optional leader sequence at the amino-terminus (N- terminus) of the CAR fusion protein.
  • the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g ., a scFv) during cellular processing and localization of the CAR to the cellular membrane.
  • a CAR that comprises an antigen binding domain (e.g., a scFv, or TCR) that binds to a specific tumor antigen X, such as those described herein, is also referred to as XCAR or CARX.
  • a CAR that comprises an antigen binding domain that binds to CD 19 is referred to as CD19CAR or CAR 19.
  • signaling domain refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen.
  • Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.
  • antibody fragment refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.
  • the portion of a CAR comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et ah, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et ah, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et ah, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et ah, 1988, Science 242:423-426).
  • the antigen binding domain of a CAR comprises an antibody fragment.
  • the CAR comprises an antibody fragment that comprises a scFv.
  • an antibody molecule refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence.
  • the term “antigen binding domain” or “antibody molecule” encompasses antibodies and antibody fragments.
  • an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • a multispecific antibody molecule is a bispecific antibody molecule.
  • a bispecific antibody has specificity for no more than two antigens.
  • a bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the portion of the CAR of the invention comprising an antigen binding domain may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or bispecific antibody (Harlow et ah, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY ; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • scFv single chain antibody
  • humanized antibody or bispecific antibody
  • the antigen binding domain of a CAR composition of the invention comprises an antibody fragment.
  • the CAR comprises an antibody fragment that comprises a scFv.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations.
  • Kappa (K) and lambda (l) light chains refer to the two major antibody light chain isotypes.
  • CDR complementarity determining region
  • HCDR1, HCDR2, and HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, and LCDR3 three CDRs in each light chain variable region
  • the precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs correspond to the amino acid residues that are part of a Rabat CDR, a Chothia CDR, or both.
  • the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.
  • recombinant antibody refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • antigen refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
  • Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically
  • xenogeneic refers to any material derived from an animal of a different species.
  • “Derived from” as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site- directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g ., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • stimulation refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR.
  • a stimulatory molecule e.g., a TCR/CD3 complex or CAR
  • its cognate ligand or tumor antigen in the case of a CAR
  • Stimulation can mediate altered expression of certain molecules.
  • the term “stimulatory molecule,” refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate activation of the immune cell in a stimulatory way for at least some aspect of the immune cell signaling pathway.
  • the signal is a primary signal that is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or IT AM.
  • IT AM containing cytoplasmic signaling sequence includes, but is not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta , CD3 epsilon, , CD79a, CD79b, DAP10, and DAP12.
  • the intracellular signaling domain in any one or more CARS of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-zeta.
  • the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO:9 (mutant CD3 zeta), or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • the primary signaling sequence of CD3-zeta is the sequence as provided in SEQ ID NO: 10 (wild-type human CD3 zeta), or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface.
  • MHC's major histocompatibility complexes
  • T-cells may recognize these complexes using their T-cell receptors (TCRs).
  • TCRs T-cell receptors
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain can generate a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell.
  • immune effector function e.g., in a CART cell
  • helper activity including the secretion of cytokines.
  • the intracellular signaling domain is the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • the intracellular signaling domain can comprise a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor
  • a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.
  • a primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or IT AM.
  • IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (“ICOS”), FceRI, and CD66d, CD32, DAP10, and DAP12.
  • zeta or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein provided as GenBank Acc. No. BAG36664.1, or the equivalent residues from a non human species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation.
  • the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc.
  • the “zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO:9. In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO: 10.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Costimulatory molecules include, but are not limited to MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signalling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD 8 alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4,
  • CD 19a and a ligand that specifically binds with CD83.
  • a costimulatory intracellular signaling domain refers to an intracellular portion of a costimulatory molecule.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • 4- IBB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non human species, e.g., mouse, rodent, monkey, ape and the like; and a “4- IBB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • the “4- IBB costimulatory domain” is the sequence provided as SEQ ID NO:7 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.
  • Immuno effector cell refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • Immuno effector function or immune effector response refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell.
  • an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell.
  • primary stimulation and co-stimulation are examples of immune effector function or response.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • the depletion can be a complete or partial depletion of the cell, protein, or macromolecule. In an embodiment, the depletion is at least a 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
  • enriched refers to the increase of the level or amount of a cell, a protein, or macromolecule in a sample after a process, e.g., a selection step, e.g., a positive selection, is performed.
  • the enrichment can be a complete or partial enrichment of the cell, protein, or macromolecule.
  • the enrichment is at least 1%, e.g., at least 1-200%, e.g., at least 1-10, 10-20, 20-30, 30-50, 50-70, 70-90, 90-110, 110-130, 130-150, 150-170, or 170-200% increase of the level or amount of a cell, a protein, or macromolecule, as compared to the level or amount of the cell, protein or macromolecule in a reference sample.
  • the enrichment is at least 5%, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% increase of the level or amount of a cell, a protein, or macromolecule, as compared to the level or amount of the cell, protein or macromolecule in a reference sample.
  • the enrichment is at least 1.1 fold, e.g., 1.1-200 fold, e.g., 1.1-10, 10-20, 20-30, 30-50, 50-70, 70-90, or 90-100 fold increase of the level or amount of a cell, a protein, or macromolecule, as compared to the level or amount of the cell, protein or macromolecule in a reference sample.
  • the reference sample can be a same sample, e.g., the sample before the process was performed.
  • the same sample refers to the sample on which the enrichment is subsequently performed, e.g., a pre-enrichment population, e.g., a starting population.
  • the reference sample can be a different sample, e.g., a sample on which the process is not performed.
  • encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g ., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “transfer vector” includes an autonomously replicating plasmid or a vims.
  • the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids ( e.g ., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody /antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Fully human refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of a DNA or RNA thereof, and polymers thereof in either single- or double-stranded form.
  • nucleic acid includes a gene, cDNA or an mRNA.
  • the nucleic acid molecule is synthetic (e.g., chemically synthesized) or recombinant. Unless specifically limited, the term encompasses nucleic acids containing analogues or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et ah, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et ah, Mol. Cell. Probes 8:91-98 (1994)).
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • promoter refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • flexible polypeptide linker or “linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
  • the flexible polypeptide linkers include, but are not limited to, (Gly4 Ser)4 (SEQ ID NO:27) or (Gly4 Ser)3 (SEQ ID NO:28).
  • the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:29). Also included within the scope of the invention are linkers described in WO2012/138475, incorporated herein by reference).
  • a 5' cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m 7 G cap) is a modified guanine nucleotide that has been added to the “front” or 5' end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other.
  • RNA polymerase Shortly after the start of transcription, the 5' end of the mRNA being synthesized is bound by a cap- synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • in vitro transcribed RNA refers to RNA, e.g., mRNA, that has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
  • the polyA is between 50 and 5000 (SEQ ID NO: 30), e.g., greater than 64, e.g., greater than 100, e.g., greater than 300 or 400 poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
  • adenosine residues are added to the free 3' end at the cleavage site.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • Apheresis is the process in which whole blood is removed from an individual, separated into select components, and the remainder returned to circulation.
  • separation of blood components centrifugal and non-centrifugal.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and/or duration of a proliferative disorder, or the amelioration of one or more symptoms (e.g., one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR of the invention).
  • the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient.
  • the terms “treat”, “treatment” and “treating” -refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
  • subject is intended to include living organisms in which an immune response can be elicited ( e.g ., mammals, human). In one embodiment, the subject is a patient.
  • a “substantially purified” cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • tumor antigen or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
  • the tumor antigen is derived from a cancer including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “specifically binds,” refers to an antibody, or a ligand, which recognizes and binds with a cognate binding partner protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
  • this disclosure provides a method for determining, e.g., predicting, a lesion-level treatment response to a chimeric antigen receptor (CAR) therapy, e.g., a CAR 19 therapy, based, e.g., on deep-learning (DL) image analysis.
  • the method further comprises a rule-based reasoning methodology for patient-level response prediction.
  • the disclosure provides a system and a non-transitory computer-readable medium for determining a lesion-level response to a CAR therapy, e.g., a CAR19 therapy.
  • a CAR therapy e.g., a CAR19 therapy.
  • the medical image-based approach disclosed herein to determine personalized response prediction to CAR T-cell therapies has certain unique advantages including: (1) the use of pre existing diagnostic imaging data sets previously acquired for clinical purposes; (2) the lack of invasiveness; (3) the extraction of regional phenotypic information from disease and extra disease sites throughout the body that may be heterogeneous; and (4) production-mode efficiency.
  • the method for determining, e.g., predicting, a lesion-level treatment response comprises: acquiring, e.g., receiving, an image of a lesion of a subject, e.g., a subject having or at risk of having a lymphoma (“acquired image”); and processing the image with a neural network (“processed image”).
  • the neural network outputs a classification result indicating the lesion- level treatment response to the CAR 19 therapy.
  • acquiring, e.g., receiving, an image of a lesion of a subject comprises acquiring at least one, two, three, four, five, six, seven, eight, nine or ten images.
  • the acquired image comprises a plurality of images, e.g., at least two, three, four, five, six, seven, eight, nine or ten images of different views of the same lesion in the subject.
  • the acquired image comprises a sagittal view, a coronal view, a transverse view, a longitudinal view, or a combination thereof, of the same lesion.
  • the acquired image is obtained by at least one imaging modality chosen from: computed tomography (CT) (e.g., diagnostic CT (dCT), low dose CT (/CT)), positron emission tomography (PET), magnetic resonance imaging (MRI), single-photon emission computerized tomography (SPECT), PET/CT, PET/MRI, SPECT/CT, /CT/PET, or a combination thereof.
  • CT computed tomography
  • PET diagnostic CT
  • MRI magnetic resonance imaging
  • SPECT single-photon emission computerized tomography
  • PET/CT positron emission tomography
  • PET/CT single-photon emission computerized tomography
  • PET/CT single-photon emission computerized tomography
  • PET/CT single-photon emission computerized tomography
  • SPECT single-photon emission computerized tomography
  • PET/CT positron emission tomography
  • PET/CT single-photon emission computerized tomography
  • SPECT single-photon emission computerized tomography
  • the acquired image is obtained by PET/CT. In some embodiments, the acquired image is obtained by PET/MRI. In some embodiments, the acquired image is obtained by SPECT/CT. In some embodiments, the acquired image is obtained by /CT/PET.
  • the acquired image is a pre-treatment image, e.g., prior to a CAR 19 therapy.
  • the acquired image is a post-treatment image, e.g., after a CAR19 therapy and/or a different therapy (e.g., chemotherapy or radiotherapy).
  • a CAR19 therapy e.g., chemotherapy or radiotherapy.
  • a different therapy e.g., chemotherapy or radiotherapy.
  • the method of predicting a lesion-level treatment response and/or a patient- level response disclosed herein can be used in a method of evaluating, or predicting the responsiveness of a subject having, or at risk of having a lymphoma, to a CAR19 therapy.
  • the method of predicting a lesion-level treatment response and/or a patient-level response can also be used in a method of treating a subject having, or at risk of having a lymphoma wherein the method comprises administering to the subject an effective amount of a CAR19 therapy.
  • the method comprises determining, e.g., predicting, a lesion-level treatment response to the CAR19 therapy with a neural network.
  • said determination comprises acquiring, e.g., receiving, an image of a lesion of the subject (“acquired image”); and processing the image with a neural network (“processed image”), wherein the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • the method further comprises a patient-level response prediction.
  • the patient-level response prediction comprises a rule-based reasoning method.
  • the patient-level response prediction comprises an All rule or a Majority rule.
  • the patient-level response prediction comprises an All rule.
  • a subject is classified as a responder or non-responder according to the All rule.
  • a responder in the All rule of the patient-level response prediction is a subject in whom all evaluated lesions have responded, or are predicted to respond to the CAR 19 therapy.
  • a non-responder in the All rule of the patient-level response prediction is a subject in whom at least one evaluated lesion has not responded, or is predicted not to respond to the CAR 19 therapy.
  • the patient-level response prediction comprises a Majority rule.
  • a subject is classified as a responder or non-responder according to the Majority rule.
  • a responder in the Majority rule of the patient-level response prediction is a subject in whom a majority of the evaluated lesions (e.g., based on a % threshold, e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have responded, or are predicted to respond to the CAR19 therapy.
  • a non-responder in the Majority rule of the patient-level response prediction is a subject in whom a majority of the evaluated lesions have not responded (e.g., based on a % threshold, e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have not responded, or are predicted not to respond to the CAR19 therapy.
  • a % threshold e.g., at least or greater than 60% (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater) of all evaluated lesions from the same subject) have not responded, or are predicted not to respond to the CAR19 therapy.
  • a responder according to the patient-level response prediction is a subject who has, or has shown a clinical response, e.g., a detectable clinical response, e.g., a complete response or a partial response.
  • a non-responder according to the patient-level response prediction is a subject who does not have, or has not shown a clinical response, e.g., does not have a detectable clinical response, e.g., has progressive disease.
  • Prognostic tools or patient stratification tools can also be used to classify patients as responders or non-responders.
  • IPI International Prognostic Index
  • FLIPI follicular lymphoma international prognostic index
  • IPI International Prognostic Index
  • DLBCL non-Hodgkin’s lymphoma
  • one point is assigned for each of the following risk factors: age > 60 years; Stage III or IV disease; elevated serum LDH levels; ECOG/Zubrod performance status of 2, 3, or 4; or more than 1 extranodal site with disease involvement.
  • the sum of the points correlates with risk groups as follows: 0-1 points correlates with low risk groups; 2 points correlates with low-intermediate risk groups; 3 points correlates with high-intermediate risk groups; and 4-5 points correlates with high risk groups.
  • the IPI index is disclosed and described in detail in the article titled “A predictive model for aggressive non-Hodgkin’s lymphoma” published in the New England Journal of Medicine (1993) Volume 329(14) pp. 987-94, the entire contents of which is incorporated by reference in its entirety.
  • FLIPI follicular lymphoma international prognostic index
  • a responder according to the patient-level response prediction is a subject who has, or has shown a clinical response, e.g., a detectable clinical response, e.g., a complete response or a partial response.
  • a responder according to the patient-level response prediction corresponds to a patient having a low score in a clinical stratification tool, e.g., International Prognostic Index (IPI) for DLBCL or follicular lymphoma international prognostic index (FLIPI) for follicular lymphoma.
  • IPI International Prognostic Index
  • FLIPI international prognostic index
  • the responder according to the patient- level response prediction has an IPI score of ⁇ 2.
  • the responder according to the patient- level response prediction has a FLIPI score of ⁇ 2.
  • a non-responder according to the patient-level response prediction is a subject who does not have, or has not shown a clinical response, e.g., does not have a detectable clinical response, e.g., has progressive disease.
  • a non-responder according to the patient-level response prediction corresponds to a subject having a high score in a clinical stratification tool, e.g., International Prognostic Index (IPI) for DLBCL and follicular lymphoma international prognostic index (FLIPI) for follicular lymphoma.
  • IPI International Prognostic Index
  • FLIPI international prognostic index
  • the non-responder according to the patient- level response prediction has an IPI score of > 2.
  • the non-responder according to the patient-level response prediction has a FLIPI score of > 2.
  • FIG. 8 depicts a block diagram of a distributed computer system 800, in which various aspects and functions discussed herein may be practiced.
  • the distributed computer system 800 may include one or more computer systems.
  • the distributed computer system 800 includes three computer systems 802, 804, 806.
  • the computer systems 802, 804 and 806 are interconnected by, and may exchange data through, a communication network 808.
  • the network 808 may include any communication network through which computer systems may exchange data.
  • the computer systems 802, 804, and 806 and the network 808 may use various methods, protocols and standards including, among others, token ring, Ethernet, Wireless Ethernet, Bluetooth, radio signaling, infra-red signaling, TCP/IP, UDP, HTTP, FTP, SNMP, SMS, MMS, SS7, JSON, XML, REST, SOAP, CORBA HOP, RMI, DCOM and Web Services.
  • the functions and operations discussed for processing image data according to any method disclosed herein can be executed on computer systems 802, 804 and 806 individually and/or in combination.
  • the computer systems 802, 804, and 806 support, for example, participation in a collaborative network.
  • a single computer system e.g., 802 can process the image data.
  • the computer systems 802, 804 and 806 may include personal computing devices such as cellular telephones, smart phones, tablets, “fablets,” etc., and may also include desktop computers, laptop computers, etc.
  • computer system 802 is a personal computing device specially configured to execute the processes and/or operations discussed herein.
  • the computer system 802 includes at least one processor 810 (e.g., a single core or a multi-core processor), a memory 812, a bus 814, input/output interfaces (e.g., 816) and storage 818.
  • the processor 810 which may include one or more microprocessors or other types of controllers, can perform a series of instructions that manipulate data.
  • the processor 810 is connected to other system components, including a memory 812, by an interconnection element (e.g., the bus 814).
  • the memory 812 and/or storage 818 may be used for storing programs and data during operation of the computer system 802.
  • the memory 812 may be a relatively high performance, volatile, random access memory such as a dynamic random access memory (DRAM) or static memory (SRAM).
  • the memory 812 may include any device for storing data, such as a disk drive or other non-volatile storage device, such as flash memory, solid state, or phase-change memory (PCM).
  • the functions and operations discussed with respect to image data processing according to any method disclosed herein can be embodied in an application that is executed on the computer system 802 from the memory 812 and/or the storage 818.
  • the application can be made available through an “app store” for download and/or purchase.
  • computer system 702 can be specially configured to execute the processing an image according to any method disclosed herein.
  • Computer system 802 also includes one or more interfaces 816 such as input devices (e.g., camera for capturing images), output devices and combination input/output devices.
  • the output devices may include a display for outputting a graphical user interface (GUI), which a user may interact with in order to carry out the methods disclosed herein.
  • GUI graphical user interface
  • the interfaces 816 may receive input, provide output, or both.
  • the storage 818 may include a computer-readable and computer- writeable nonvolatile storage medium in which instructions are stored that define a program to be executed by the processor.
  • the storage system 818 also may include information that is recorded, on or in, the medium, and this information may be processed by the application.
  • a medium that can be used with various embodiments may include, for example, optical disk, magnetic disk or flash memory, SSD, among others. Further, aspects and embodiments are not to a particular memory system or storage system.
  • the computer system 802 may include an operating system that manages at least a portion of the hardware components (e.g., input/output devices, touch screens, cameras, etc.) included in computer system 802.
  • One or more processors or controllers, such as processor 810 may execute an operating system which may be, among others, a Windows-based operating system (e.g., Windows NT, ME, XP, Vista, 7, 8, 10, or RT) available from the Microsoft Corporation, an operating system available from Apple Computer (e.g., MAC OS, including System X), one of many Linux-based operating system distributions (for example, the Enterprise Linux operating system available from Red Hat Inc.), a Solaris operating system available from Sun Microsystems, or a UNIX operating systems available from various sources.
  • a Windows-based operating system e.g., Windows NT, ME, XP, Vista, 7, 8, 10, or RT
  • Apple Computer e.g., MAC OS, including System X
  • Linux-based operating system distributions for example, the Enterprise Linux
  • the processor and operating system together define a computing platform on which applications (e.g., “apps” available from an “app store”) may be executed.
  • applications e.g., “apps” available from an “app store”
  • various functions for generating and manipulating images may be implemented in a non- programmed environment (for example, documents created in HTML, XML or other format that, when viewed in a window of a browser program, render aspects of a graphical-user interface or perform other functions).
  • various embodiments in accord with aspects of the present invention may be implemented as programmed or non-programmed components, or any combination thereof.
  • Various embodiments may be implemented in part as MATLAB functions, scripts, and/or batch jobs.
  • the invention is not limited to a specific programming language and any suitable programming language could also be used.
  • a transfer learning program or incremental learning program implementing one or more methods disclosed herein is implemented on computer system 802 and/or one or more of computer systems 804 and 806.
  • System 802 may be, for example, a personal computer (PC) executing the program in MATLAB 2018b under Ubuntu 16.04 OS.
  • the memory 812 of system 802 may include 64 GB RAM.
  • the processor 810 of system 802 may be one of four Intel Core G7 CPUs included in system 802. Additionally, bus 814 of system 802 may be connected to two Nvidia 1080T ⁇ graphics processing unit (GPU) cards with 22 GB GPU RAM in total.
  • GPU graphics processing unit
  • the one or more of computer systems 804 and 806 may each be another PC with a respective memory of 24 GB RAM, processors including four Intel Core G7 CPUs, and one Nvidia TITAN V GPU card with 12 GB GPU RAM connected to a respective bus of the one or more of computer systems 804 and 806.
  • various embodiments of systems implementing the transfer learning program or incremental learning program may utilize a different operating system, use a different type or number of processors, a different amount of system memory, or a different GPU.
  • the program may be implemented entirely on a single computer system.
  • computer system 802 is shown by way of example as one type of computer system upon which various functions for processing image data according to any method disclosed herein may be practiced, aspects and embodiments are not limited to being implemented on the computer system, shown in FIG. 8. Various aspects and functions may be practiced on one or more computers or similar devices having different architectures or components than that shown in FIG. 8.
  • FIG. 9 is flowchart depicting the transfer learning method 900.
  • the method 900 includes embodiments described by the transfer learning shown in FIG. 3 as well as the enumerated embodiments and examples discussed herein.
  • the method 900 may be implemented, for example, on one or more computers in distributed computer system 800. However, aspects and embodiments are not limited to being implemented on only computer system 802 or any subset of computer systems in the distribute computer system 800. Any computer system suitable for deep learning applications may be utilized, preferably where the computer system includes one or more dedicated GPUs.
  • image data for a given scenario such as the 1 VOI slice scenario
  • the image data may include any one or more of the input scenarios shown in the transfer learning or incremental learning in FIG. 3 and described in the enumerated embodiments and examples. In other embodiments more than one scenario may be combined together to form a new scenario.
  • step S904 a pre-trained neural network, or any other network, that has been modified for a new task is loaded as described herein.
  • the new convolutional neural network from FIG. 2 is loaded.
  • a batch size and epoch may be specified.
  • step S906 transfer learning is performed on the modified network using the image data for a given imaging modality, such as dCT, /CT, PET, or any other medical imaging modality.
  • a given imaging modality such as dCT, /CT, PET, or any other medical imaging modality.
  • each iteration of step S906 uses the same network loaded in step S904.
  • step S908 the trained, modified neural network from step S906 is output and stored for use with any prediction task disclosed herein.
  • step S910 a determination is made if there is a remaining imaging modality to utilize for the given scenario. If so, step S906 is repeated to create an additional trained network to output and store at step S908. If not, the method proceeds to step S912.
  • step S912 a determination is made if another scenario for a same or different imaging modality from that already processed by step S902 remains. If so, steps S902, S904, S906, S908, and S910 are repeated. If not, method 900 ends and each of the one or more stored neural networks in step S908 is usable for a prediction task according to any of enumerated embodiments and examples discussed herein.
  • the image data for all scenarios to be processed may be input at step S902, and step S912 proceeds to step S904 instead of step S902 in the event another scenario remains.
  • the steps or portions of the steps may be performed in a different order to achieve the same result(s) produced at step S908.
  • FIG. 10 is flowchart depicting the incremental learning method 1000.
  • the method 1000 includes embodiments described by the transfer learning and incremental learning in FIG. 3 as well as the enumerated embodiments and examples discussed herein.
  • the method 1000 may be implemented, for example, on one or more computers in distributed computer system 800. However, aspects and embodiments are not limited to being implemented on only computer system 802 or any subset of computer systems in the distribute computer system 800. Any computer system suitable for deep learning applications may be utilized, preferably where the computer system includes one or more dedicated GPUs.
  • image data is received as described in the enumerated embodiments and examples discussed herein.
  • the image data may include any one or more of the input scenarios shown in the transfer learning or incremental learning in FIG. 3 and described in the enumerated embodiments and examples. In other embodiments more than one scenario may be combined together to form a new scenario.
  • step S1004 a pre-trained neural network, or any other network, that has been modified for a new task is loaded as described herein.
  • the new convolutional neural network from FIG. 2 is loaded.
  • a batch size and epoch may be specified.
  • steps S1002 and S1004 are performed in the opposite order.
  • step S1006 transfer learning is performed on the modified network from step S1004 using the image data for a first imaging modality, such as dCT, and a given scenario, such as the 1 VOI slice scenario.
  • a first imaging modality such as dCT
  • a given scenario such as the 1 VOI slice scenario.
  • each iteration of step S1006 uses the same network loaded in step S1004.
  • step S1008 the network trained in step S1006 is re-trained for a second imaging modality, such as /CT.
  • a second imaging modality such as /CT.
  • the first imaging modality and second imaging modality may be the same. In another embodiment the first imaging modality and second imaging modality may be different.
  • step S1010 the re-trained network from step S1008 is output and stored for use with any prediction task disclosed herein.
  • step S1012 a determination is made if another second imaging modality, such as PET, exists for the given scenario processed by steps S1006 and S1008. If so, step S1008 is repeated. If not, the method proceeds to step S1014.
  • another second imaging modality such as PET
  • step S1014 a determination is made if another scenario for the first modality remains. If so, steps S1006, S1008, S1010, and S1012 are repeated to produce one or more new re-trained networks to store. If not, the method proceeds to step S1016.
  • step S1016 a determination is made if another first modality remains. If so, steps S1006, S1008, S1010, S1012, and S1014 are repeated to produce one or more new re-trained networks to store. If not, method 1000 ends and each of the one or more stored neural networks in step S1010 is usable for a prediction task according to any of enumerated embodiments and examples discussed herein. In various embodiments, the steps or portions of the steps may be performed in a different order to achieve the same result(s) produced at step S1010.
  • the present invention provides immune effector cells (e.g ., T cells, NK cells) that are engineered to contain one or more CARs that direct the immune effector cells to cancer. This is achieved through an antigen binding domain on the CAR that is specific for a cancer associated antigen.
  • cancer associated antigens tumor antigens
  • MHC major histocompatibility complex
  • an immune effector cell e.g., obtained by a method described herein, can be engineered to contain a CAR that targets the CD 19 antigen.
  • a multispecific antibody molecule is a bispecific antibody molecule.
  • a bispecific antibody has specificity for no more than two antigens.
  • a bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein).
  • the first and second epitopes overlap.
  • the first and second epitopes do not overlap.
  • first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein).
  • a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope.
  • a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope.
  • a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope.
  • a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope.
  • the antibody molecule is a multi-specific (e.g., a bispecific or a trispecific) antibody molecule.
  • Protocols for generating bispecific or heterodimeric antibody molecules, and various configurations for bispecific antibody molecules, are described in, e.g., paragraphs 455-458 of WO2015/142675, filed March 13, 2015, which is incorporated by reference in its entirety.
  • the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence, e.g., a scFv, which has binding specificity for CD 19, e.g., comprises a scFv as described herein, or comprises the light chain CDRs and/or heavy chain CDRs from a scFv described herein, and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope on a different antigen.
  • a first immunoglobulin variable domain sequence e.g., a scFv
  • CD 19 comprises a scFv as described herein, or comprises the light chain CDRs and/or heavy chain CDRs from a scFv described herein
  • a second immunoglobulin variable domain sequence that has binding specificity for a second epitope on a different antigen.
  • the antibodies and antibody fragments of the present invention e.g., the antibodies and antibody fragments of the present invention.
  • CD 19 antibodies and fragments can be grafted to one or more constant domain of a T cell receptor (“TCR”) chain, for example, a TCR alpha or TCR beta chain, to create a chimeric TCR.
  • TCR T cell receptor
  • an scFv as disclosed herein can be grafted to the constant domain, e.g., at least a portion of the extracellular constant domain, the transmembrane domain and the cytoplasmic domain, of a TCR chain, for example, the TCR alpha chain and/or the TCR beta chain.
  • an antibody fragment for example a VL domain as described herein, can be grafted to the constant domain of a TCR alpha chain
  • an antibody fragment for example a VH domain as described herein
  • a VL domain may be grafted to the constant domain of the TCR beta chain
  • a VH domain may be grafted to a TCR alpha chain
  • the CDRs of an antibody or antibody fragment may be grafted into a TCR alpha and/or beta chain to create a chimeric TCR.
  • the LCDRs disclosed herein may be grafted into the variable domain of a TCR alpha chain and the HCDRs disclosed herein may be grafted to the variable domain of a TCR beta chain, or vice versa.
  • Such chimeric TCRs may be produced, e.g., by methods known in the art (For example, Willemsen RA et al, Gene Therapy 2000; 7: 1369-1377; Zhang T et al, Cancer Gene Ther 2004; 11: 487-496; Aggen et al, Gene Ther. 2012 Apr;19(4):365-74).
  • the antigen binding domain comprises a non-antibody scaffold, e.g., a fibronectin, ankyrin, domain antibody, lipocalin, small modular immuno-pharmaceutical, maxybody, Protein A, or affilin.
  • the non-antibody scaffold has the ability to bind to target antigen on a cell.
  • the antigen binding domain is a polypeptide or fragment thereof of a naturally occurring protein expressed on a cell.
  • the antigen binding domain comprises a non-antibody scaffold.
  • a wide variety of non-antibody scaffolds can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to the target antigen on a target cell.
  • Non-antibody scaffolds include: fibronectin (Novartis, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc., Mountain View, CA), Protein A (Affibody AG, Sweden), and affilin (gamma- crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).
  • the antigen binding domain comprises the extracellular domain, or a counter-ligand binding fragment thereof, of molecule that binds a counterligand on the surface of a target cell.
  • the immune effector cells can comprise a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds specifically to a tumor antigen, e.g., a tumor antigen described herein, and an intracellular signaling domain.
  • the intracellular signaling domain can comprise a costimulatory signaling domain and/or a primary signaling domain, e.g., a zeta chain.
  • the methods described herein can include transducing a cell, e.g., from the population of T regulatory -depleted cells, with a nucleic acid encoding a CAR, e.g., a CAR described herein.
  • a CAR comprises a scFv domain, wherein the scFv may be preceded by an optional leader sequence such as provided in SEQ ID NO: 1, and followed by an optional hinge sequence such as provided in SEQ ID NO:2 or SEQ ID NO:36 or SEQ ID NO:38, a transmembrane region such as provided in SEQ ID NO:6, an intracellular signalling domain that includes SEQ ID NO:7 or SEQ ID NO: 16 and a CD3 zeta sequence that includes SEQ ID NO:9 or SEQ ID NO: 10, e.g., wherein the domains are contiguous with and in the same reading frame to form a single fusion protein.
  • an optional leader sequence such as provided in SEQ ID NO: 1
  • an optional hinge sequence such as provided in SEQ ID NO:2 or SEQ ID NO:36 or SEQ ID NO:38
  • a transmembrane region such as provided in SEQ ID NO:6
  • an intracellular signalling domain that includes SEQ ID NO:7 or SEQ ID
  • an exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain ( e.g ., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein).
  • an optional leader sequence e.g., a leader sequence described herein
  • an extracellular antigen binding domain e.g., an antigen binding domain described herein
  • a hinge e.g., a hinge region described herein
  • a transmembrane domain e.g ., a transmembrane domain described herein
  • an intracellular stimulatory domain e.g., an intracellular stimulatory domain described herein
  • an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein) and/or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).
  • an optional leader sequence e.g., a leader sequence described herein
  • an extracellular antigen binding domain e.g., an antigen binding domain described herein
  • a hinge e.g., a hinge region described herein
  • a transmembrane domain e.g., a transmembrane domain described herein
  • an intracellular costimulatory signaling domain e.g., a costim
  • An exemplary leader sequence is provided as SEQ ID NO: 1.
  • An exemplary hinge/spacer sequence is provided as SEQ ID NO: 2 or SEQ ID NO:36 or SEQ ID NO:38.
  • An exemplary transmembrane domain sequence is provided as SEQ ID NO:6.
  • An exemplary sequence of the intracellular signaling domain of the 4- IBB protein is provided as SEQ ID NO: 7.
  • An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO: 16.
  • An exemplary CD3zeta domain sequence is provided as SEQ ID NO: 9 or SEQ ID NO: 10.
  • the immune effector cell comprises a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen binding domain, wherein the sequence is contiguous with and in the same reading frame as the nucleic acid sequence encoding an intracellular signaling domain.
  • An exemplary intracellular signaling domain that can be used in the CAR includes, but is not limited to, one or more intracellular signaling domains of, e.g., CD3-zeta, CD28, CD27, 4- IBB, and the like.
  • the CAR can comprise any combination of CD3-zeta, CD28, 4- IBB, and the like.
  • nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the nucleic acid molecule, by deriving the nucleic acid molecule from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • the nucleic acid of interest can be produced synthetically, rather than cloned.
  • Nucleic acids encoding a CAR can be introduced into the immune effector cells using, e.g., a retroviral or lentiviral vector construct. Nucleic acids encoding a CAR can also be introduced into the immune effector cell using, e.g., an RNA construct that can be directly transfected into a cell.
  • a method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”) (e.g., a 3’ and/or 5’ UTR described herein), a 5’ cap (e.g., a 5’ cap described herein) and/or Internal Ribosome Entry Site (IRES) (e.g., an IRES described herein), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO: 35) (e.g., described in the Examples, e.g., SEQ ID NO:35).
  • RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the CAR.
  • an RNA CAR vector is transduced into a cell, e.g., a T cell by electroporation.
  • a plurality or population of the immune effector cells include a nucleic acid encoding a CAR that comprises a target- specific binding element otherwise referred to as an antigen binding domain.
  • the choice of binding element depends upon the type and number of ligands that define the surface of a target cell.
  • the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
  • examples of cell surface markers that may act as ligands for the antigen binding domain in a CAR described herein include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells.
  • the portion of the CAR comprising the antigen binding domain comprises an antigen binding domain that targets a tumor antigen, e.g., a tumor antigen described herein.
  • the antigen binding domain can be any domain that binds to the antigen including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), or a fragment there of, e.g., single chain TCR, and the like.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VHH variable domain of camelid derived nanobody
  • an alternative scaffold known in the art to function as antigen binding domain such as a recombinant fibronectin domain, a T cell receptor (TCR), or a fragment there of,
  • the antigen binding domain comprises an anti-CD 19 antibody, or fragment thereof, e.g., an scFv.
  • the antigen binding domain comprises a variable heavy chain and a variable light chain listed in Table 1.
  • the linker sequence joining the variable heavy and variable light chains can be, e.g., any of the linker sequences described herein, or alternatively, can be GSTSGSGKPGSGEGSTKG (SEQ ID NO: 104).
  • the antigen binding domain comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any heavy chain binding domain amino acid sequences listed in Table 1 or Table 2. In embodiments, the antigen binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. In embodiments, the antigen binding domain comprises a LC CDR1, a LC CDR2, and a LC CDR3 of any light chain binding domain amino acid sequences listed in Table 1 or Table 2.
  • the antigen binding domain comprises one, two or all of LC CDR1, LC CDR2, and LC CDR3 of any light chain binding domain amino acid sequences listed in Table 1, and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of any heavy chain binding domain amino acid sequences listed in Table 1.
  • the antigen binding domain comprises one, two or all of LC CDR1, LC CDR2, and LC CDR3 of any light chain binding domain amino acid sequences listed in Table 2, and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of any heavy chain binding domain amino acid sequences listed in Table 2.
  • CD 19 CAR e.g., the CD 19 antigen binding domain of any known CD 19 CAR
  • the CAR T cell that specifically binds to CD19 has the USAN designation TISAGENLECLEUCEL-T.
  • CTL019 is made by a gene modification of T cells is mediated by stable insertion via transduction with a self-inactivating, replication deficient Lentiviral (LV) vector containing the CTL019 transgene under the control of the EF-1 alpha promoter.
  • LV replication deficient Lentiviral
  • CTL019 can be a mixture of transgene positive and negative T cells that are delivered to the subject on the basis of percent transgene positive T cells.
  • the CAR-expressing cells can specifically bind to human CD 19, e.g., can include a CAR molecule, or an antigen binding domain (e.g., a humanized antigen binding domain) according to Table 3 of WO2014/153270, incorporated herein by reference.
  • an antigen binding domain e.g., a humanized antigen binding domain
  • the anti-tumor antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv).
  • the anti-a cancer associate antigen as described herein binding domain is a Fv, a Fab, a (Fab')2, or a bi-functional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).
  • the antibodies and fragments thereof of the invention binds a cancer associate antigen as described herein protein with wild-type or enhanced affinity.
  • scFvs can be prepared according to a method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl.
  • ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers.
  • the scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition.
  • the linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site.
  • linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos.
  • An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions.
  • the linker sequence may comprise any naturally occurring amino acid.
  • the linker sequence comprises amino acids glycine and serine.
  • the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO:25).
  • the linker can be (Gly 4 Ser) 4 (SEQ ID NO:27) or (Gly4Ser)3(SEQ ID NO:28). Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • the antigen binding domain is a T cell receptor (“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR).
  • TCR T cell receptor
  • scTCR single chain TCR
  • Methods to make such TCRs are known in the art. See, e.g., Willemsen RA et al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (references are incorporated herein by its entirety).
  • scTCR can be engineered that contains the Va and nb genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is very useful to cancer associated target that itself is intracellular, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC.
  • a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region).
  • the transmembrane domain is one that is associated with one of the other domains of the CAR.
  • the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CART.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target.
  • a transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIR2DS2, 0X40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, IT GAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD
  • the CAR molecule comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain comprises a sequence of SEQ ID NO: 6.
  • the transmembrane domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 6, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 6.
  • the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen binding domain of the CAR, via a hinge, e.g., a hinge from a human protein.
  • the hinge can be a human Ig (immunoglobulin) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:2.
  • the transmembrane domain comprises (e.g., consists of) a transmembrane domain of SEQ ID NO: 6.
  • the hinge or spacer comprises an IgG4 hinge.
  • the hinge or spacer comprises a hinge of the amino acid sequence ES KY GPPCPPCPAPEFLGGPS VFLFPPKPKDTLMISRTPE VTC VVVD V S QEDPEV QFNW Y VDG VE VHN AKTKPREEQFN S T YR V V S VLT VLHQD WLN GKE YKC KV S NKGLPS S IEK TIS KAKGQPREPQ V YTLPPS QEEMTKN Q V S LTCLVKGF YPS DIA VE WES NGQPENN YK TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM (SEQ ID NO: 36).
  • the hinge or spacer comprises a hinge encoded by a nucleotide sequence of
  • the hinge or spacer comprises an IgD hinge.
  • the hinge or spacer comprises a hinge of the amino acid sequence RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERET KTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTG G VEEGLLERHS NGS QS QHS RLTLPRS LWN AGTS VTCTLNHPS LPPQRLM ALREP A AQ A P VKLS LNLL AS S DPPE A AS WLLCE V S GFS PPNILLMWLED QRE VNT S GFAP ARPPPQPG S TTFW AW S VLRVP APPS PQP AT YT C V V S HEDS RTLLN AS RS LEVS YVTDH (SEQ ID NO:38).
  • the hinge or spacer comprises a hinge encoded by a nucleotide sequence of
  • the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine can be found at each end of a recombinant transmembrane domain.
  • a short oligo- or polypeptide linker may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR.
  • a glycine- serine doublet provides a particularly suitable linker.
  • the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 5).
  • the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 8).
  • the hinge or spacer comprises a KIR2DS2 hinge.
  • the cytoplasmic domain or region of the CAR includes an intracellular signaling domain.
  • An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced.
  • Examples of intracellular signaling domains for use in a CAR described herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • TCR T cell receptor
  • T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).
  • a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.
  • a CAR of the invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta, e.g., a CD3-zeta sequence described herein.
  • a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
  • a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain.
  • a primary signaling domain comprises one, two, three, four or more ITAM motifs.
  • the intracellular signalling domain of the CAR can comprise the CD3-zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR of the invention.
  • the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain.
  • the costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28.
  • the intracellular domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of ICOS.
  • a costimulatory molecule can be a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood.
  • costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp30, NKp44, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, IT GAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM
  • the intracellular signaling sequences within the cytoplasmic portion of the CAR may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g ., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
  • the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains.
  • the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains are separated by a linker molecule, e.g., a linker molecule described herein.
  • the intracellular signaling domain comprises two costimulatory signaling domains.
  • the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue.
  • the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28.
  • the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4- IBB.
  • the signaling domain of 4- IBB is a signaling domain of SEQ ID NO: 7.
  • the 4- IBB costimulatory domain comprises a sequence of SEQ ID NO: 7.
  • the 4-1BB costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 7, or a sequence with at least 95%, e.g., 95-99%, identity to an amino acid sequence of SEQ ID NO:7.
  • the signaling domain of CD3-zeta is a signaling domain of SEQ ID NO: 9.
  • the CD3-zeta costimulatory domain comprises a sequence of SEQ ID NO: 9.
  • the CD3-zeta costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 9, or a sequence with at least 95%, e.g., 95-99%, identity to an amino acid sequence of SEQ ID NO:9.
  • the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD27.
  • the signaling domain of CD27 comprises an amino acid sequence of
  • the signalling domain of CD27 is encoded by a nucleic acid sequence of AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCC GCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCA GCCTATCGCTCC (SEQ ID NO: 14).
  • the CD27 costimulatory domain comprises a sequence of SEQ ID NO: 14. In one embodiment, the CD27 costimulatory domain comprises an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 20, 10 or 5 modifications (e.g., substitutions) of an amino acid sequence of SEQ ID NO: 14, or a sequence with at least 95%, e.g., 95-99%, identity to an amino acid sequence of SEQ ID NO:14.
  • the CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., to the same target or a different target (e.g., a target other than a cancer associated antigen described herein or a different cancer associated antigen described herein, e.g., CD19, CD33, CLL-1, CD34, FLT3, or folate receptor beta).
  • the second CAR includes an antigen binding domain to a target expressed the same cancer cell type as the cancer associated antigen.
  • the CAR-expressing cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different, antigen and includes an intracellular signaling domain having a primary signaling domain but not a costimulatory signaling domain.
  • a costimulatory signaling domain e.g., 4-1BB, CD28, ICOS, CD27 or OX-40
  • the CAR expressing cell comprises a first cancer associated antigen CAR that includes an antigen binding domain that binds a target antigen described herein, a transmembrane domain and a costimulatory domain and a second CAR that targets a different target antigen (e.g., an antigen expressed on that same cancer cell type as the first target antigen) and includes an antigen binding domain, a transmembrane domain and a primary signaling domain.
  • a target antigen e.g., an antigen expressed on that same cancer cell type as the first target antigen
  • the CAR expressing cell comprises a first CAR that includes an antigen binding domain that binds a target antigen described herein, a transmembrane domain and a primary signaling domain and a second CAR that targets an antigen other than the first target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and includes an antigen binding domain to the antigen, a transmembrane domain and a costimulatory signaling domain.
  • a first CAR that includes an antigen binding domain that binds a target antigen described herein, a transmembrane domain and a primary signaling domain
  • a second CAR that targets an antigen other than the first target antigen e.g., an antigen expressed on the same cancer cell type as the first target antigen
  • the disclosure features a population of CAR-expressing cells, e.g., CART cells.
  • the population of CAR-expressing cells comprises a mixture of cells expressing different CARs.
  • the population of CART cells can include a first cell expressing a CAR having an antigen binding domain to a cancer associated antigen described herein, and a second cell expressing a CAR having a different antigen binding domain, e.g., an antigen binding domain to a different a cancer associated antigen described herein, e.g., an antigen binding domain to a cancer associated antigen described herein that differs from the cancer associate antigen bound by the antigen binding domain of the CAR expressed by the first cell.
  • the population of CAR-expressing cells can include a first cell expressing a CAR that includes an antigen binding domain to a cancer associated antigen described herein, and a second cell expressing a CAR that includes an antigen binding domain to a target other than a cancer associate antigen as described herein.
  • the population of CAR-expressing cells includes, e.g., a first cell expressing a CAR that includes a primary intracellular signaling domain, and a second cell expressing a CAR that includes a secondary signaling domain.
  • the disclosure features a population of cells wherein at least one cell in the population expresses a CAR having an antigen binding domain to a cancer associated antigen described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a CAR-expressing cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., PD-1, can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response.
  • inhibitory molecules include PD-1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGF (e.g., TGF beta).
  • TGF e.g., TGF beta
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF beta, or a fragment of any of these, and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4 IBB, CD27, 0X40 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein).
  • an inhibitory molecule such as PD-1, PD-L1, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT
  • the agent comprises a first polypeptide of PD-1 or a fragment thereof, and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
  • a second polypeptide of an intracellular signaling domain described herein e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein.
  • sequences of anti-CD 19 binding domains are provided herein in Table 1.
  • Full CAR constructs can be generated using any of the antigen binding domains described in Table 1 with one or more additional CAR component provided below.
  • leader nucleic acid sequence 3 (SEQ ID NO: 128)
  • CD8 hinge amino acid sequence
  • CD8 hinge nucleic acid sequence
  • CD8 hinge nucleic acid sequence 2 (SEQ ID NO: 129)
  • CD8 transmembrane amino acid sequence
  • SEQ ID NO: 6 CD8 transmembrane (amino acid sequence)
  • CD3 zeta domain (amino acid sequence) (SEQ ID NO: 9) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR
  • CD3 zeta nucleic acid sequence
  • CD3 zeta nucleic acid sequence 2 (SEQ ID NO: 132)
  • CD3 zeta domain (amino acid sequence; NCBI Reference Sequence NM_000734.3) (SEQ ID NO:10)
  • IgG4 Hinge (nucleotide sequence) (SEQ ID NO: 37)
  • Gly/Ser SEQ ID NO:26: This sequence may encompass 1-6 "Gly Gly Gly Gly Ser" repeating units
  • GGGGS GGGGS GGGGSGGGGS GGGGS GGGGS
  • PolyA (SEQ ID NO:30): A5000 PolyA (SEQ ID NO:31): A100 PolyT (SEQ ID NO:32): T5000 PolyA (SEQ ID NO:33): A5000 PolyA (SEQ ID NO:34): A400 PolyA (SEQ ID NO:35)” A2000 ⁇ Gly/Ser (SEQ ID NO: 15): This sequence may encompass 1-10 "Gly Gly Gly Ser" repeating units
  • CD 19 CAR constructs that can be used in the methods described herein are shown in Table 3:
  • the antigen binding domain comprises a HC CDR1, a HC CDR2, and a HC CDR3 of any heavy chain binding domain amino acid sequences listed in Table 3A. In embodiments, the antigen binding domain further comprises a LC CDR1, a LC CDR2, and a LC CDR3. In embodiments, the antigen binding domain comprises a LC CDR1, a LC CDR2, and a LC CDR3 of any light chain binding domain amino acid sequences listed in Table 3B.
  • the antigen binding domain comprises one, two or all of LC CDR1, LC CDR2, and LC CDR3 of any light chain binding domain amino acid sequences listed in Table 3B, and one, two or all of HC CDR1, HC CDR2, and HC CDR3 of any heavy chain binding domain amino acid sequences listed in Table 3A.
  • the antigen binding domain (e.g., an scFv) comprises: a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a light chain variable region provided in Table 3, or a sequence with at least 95%, e.g., 95-99%, identity with an amino acid sequence of Table 3; and/or a heavy chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of a heavy chain variable region provided in Table 3, or a sequence with 95-99% identity to an amino acid sequence of Table 3.
  • a light chain variable region comprising an amino acid sequence having at least one, two or three modifications (e.g., substitutions) but not more than 30, 20 or 10 modifications (e.g., substitutions) of an amino acid sequence of
  • the CAR is a CD 19 CAR comprising an antigen binding domain which comprises an scFv amino acid sequence provided in Table 3.
  • the CD19 CAR comprises an scFv amino acid sequence of any one of SEQ ID NO: 39-51, SEQ ID NO: 144 or SEQ ID NO: 147, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity thereto.
  • the CAR is a CD 19 CAR comprising a CAR amino acid sequence provided in Table 3.
  • the CD 19 CAR comprises the amino acid sequence of any one of SEQ ID NO: 77-89, SEQ ID NO: 145 or SEQ ID NO: 146, or a sequence with at least 80%, 85%, 90%, 95%, or 99% identity thereto.
  • the CDRs are defined according to the Rabat numbering scheme, the Chothia numbering scheme, or a combination thereof.
  • the CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., to the same target (e.g., CD 19) or a different target (e.g., a target other than CD 19, e.g., a target described herein).
  • the CAR-expressing cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different, antigen and includes an intracellular signaling domain having a primary signaling domain but not a costimulatory signaling domain. Placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27, OX-40 or ICOS, onto the first CAR, and the primary signaling domain, e.g.,
  • a costimulatory signaling domain e.g., 4
  • the CAR expressing cell comprises a first CAR that includes an antigen binding domain, a transmembrane domain and a costimulatory domain and a second CAR that targets another antigen and includes an antigen binding domain, a transmembrane domain and a primary signaling domain.
  • the CAR expressing cell comprises a first CAR that includes an antigen binding domain, a transmembrane domain and a primary signaling domain and a second CAR that targets another antigen and includes an antigen binding domain to the antigen, a transmembrane domain and a costimulatory signaling domain.
  • the CAR-expressing cell comprises an XCAR described herein and an inhibitory CAR.
  • the inhibitory CAR comprises an antigen binding domain that binds an antigen found on normal cells but not cancer cells, e.g., normal cells that also express X.
  • the inhibitory CAR comprises the antigen binding domain, a transmembrane domain and an intracellular domain of an inhibitory molecule.
  • the intracellular domain of the inhibitory CAR can be an intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAF9, adenosine, and TGF (e.g ., TGF beta).
  • TGF e.g ., TGF beta
  • the antigen binding domains of the different CARs can be such that the antigen binding domains do not interact with one another.
  • a cell expressing a first and second CAR can have an antigen binding domain of the first CAR, e.g., as a fragment, e.g., an scFv, that does not form an association with the antigen binding domain of the second CAR, e.g., the antigen binding domain of the second CAR is a VHH.
  • the antigen binding domain comprises a single domain antigen binding (SDAB) molecules include molecules whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules naturally devoid of light chains, single domains derived from conventional 4-chain antibodies, engineered domains and single domain scaffolds other than those derived from antibodies. SDAB molecules may be any of the art, or any future single domain molecules. SDAB molecules may be derived from any species including, but not limited to mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. This term also includes naturally occurring single domain antibody molecules from species other than Camelidae and sharks.
  • SDAB single domain antigen binding
  • an SDAB molecule can be derived from a variable region of the immunoglobulin found in fish, such as, for example, that which is derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark.
  • NAR Novel Antigen Receptor
  • an SDAB molecule is a naturally occurring single domain antigen binding molecule known as heavy chain devoid of light chains.
  • Such single domain molecules are disclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example.
  • this variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins.
  • a VHH molecule can be derived from Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain; such VHHs are within the scope of the invention.
  • the SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, de- immunized and/or in vitro generated ( e.g ., selected by phage display).
  • cells having a plurality of chimeric membrane embedded receptors comprising an antigen binding domain that interactions between the antigen binding domain of the receptors can be undesirable, e.g., because it inhibits the ability of one or more of the antigen binding domains to bind its cognate antigen.
  • cells having a first and a second non-naturally occurring chimeric membrane embedded receptor comprising antigen binding domains that minimize such interactions are also disclosed herein.
  • nucleic acids encoding a first and a second non-naturally occurring chimeric membrane embedded receptor comprising an antigen binding domains that minimize such interactions, as well as methods of making and using such cells and nucleic acids.
  • the antigen binding domain of one of the first and the second non- naturally occurring chimeric membrane embedded receptor comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.
  • a composition herein comprises a first and second CAR, wherein the antigen binding domain of one of the first and the second CAR does not comprise a variable light domain and a variable heavy domain.
  • the antigen binding domain of one of the first and the second CAR is an scFv, and the other is not an scFv.
  • the antigen binding domain of one of the first and the second CAR comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.
  • the antigen binding domain of one of the first and the second CAR comprises a nanobody.
  • the antigen binding domain of one of the first and the second CAR comprises a camelid VHH domain.
  • the antigen binding domain of one of the first and the second CAR comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.
  • the antigen binding domain of one of the first and the second CAR comprises an scFv, and the other comprises a nanobody.
  • the antigen binding domain of one of the first and the second CAR comprises an scFv, and the other comprises a camelid VHH domain.
  • binding of the antigen binding domain of the first CAR to its cognate antigen is not substantially reduced by the presence of the second CAR.
  • binding of the antigen binding domain of the first CAR to its cognate antigen in the presence of the second CAR is at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% of binding of the antigen binding domain of the first CAR to its cognate antigen in the absence of the second CAR.
  • the antigen binding domains of the first and the second CAR when present on the surface of a cell, associate with one another less than if both were scFv antigen binding domains. In some embodiments, the antigen binding domains of the first and the second CAR, associate with one another at least 85%, 90%, 95%, 96%, 97%, 98% or 99% less than, e.g., 85%, 90%, 95%, 96%, 97%, 98% or 99% less than if both were scFv antigen binding domains.
  • the CAR-expressing cell described herein can further express another agent, e.g., an agent that enhances the activity or fitness of a CAR-expressing cell.
  • the agent can be an agent which inhibits a molecule that modulates or regulates, e.g., inhibits, T cell function.
  • the molecule that modulates or regulates T cell function is an inhibitory molecule.
  • Inhibitory molecules, e.g., PD1 can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response.
  • inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, or TGF beta.
  • an agent e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA; or e.g., an inhibitory protein or system, e.g., a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein, can be used to inhibit expression of a molecule that modulates or regulates, e.g., inhibits, T-cell function in the CAR- expressing cell.
  • an inhibitory nucleic acid e.g., a dsRNA, e.g., an siRNA or shRNA
  • an inhibitory protein or system e.g., a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN
  • the agent is an shRNA, e.g., an shRNA described herein.
  • the agent that modulates or regulates, e.g., inhibits, T-cell function is inhibited within a CAR-expressing cell.
  • a dsRNA molecule that inhibits expression of a molecule that modulates or regulates, e.g., inhibits, T-cell function is linked to the nucleic acid that encodes a component, e.g., all of the components, of the CAR.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, PD-F1, CTFA4, TIM3, FAG3, VISTA, BTFA, TIGIT, FAIR1, CD 160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAF9, adenosine, or TGF beta, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g.,
  • the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
  • PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTFA-4, ICOS, and BTFA.
  • PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75).
  • Two ligands for PD1, PD-F1 and PD-F2 have been shown to downregulate T cell activation upon binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Fatchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43).
  • PD-F1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7;
  • the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD1), can be fused to a transmembrane domain and intracellular signaling domains such as 4 IBB and CD3 zeta (also referred to herein as a PD1 CAR).
  • the PD1 CAR when used in combinations with an XCAR described herein, improves the persistence of the T cell.
  • the CAR is a PD1 CAR comprising the extracellular domain of PD1 indicated as underlined in SEQ ID NO: 105.
  • the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 105.
  • the PD1 CAR comprises the amino acid sequence provided below (SEQ ID NO: 106). pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfylnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlp ngrdfhmsvyrarrndsgtylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpaptiasqplslrp eacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkf srsadapaykq
  • the agent comprises a nucleic acid sequence encoding the PD1 CAR, e.g., the PD1 CAR described herein.
  • the nucleic acid sequence for the PD1 CAR is shown below, with the PD1 ECD underlined below in SEQ ID NO: 107 atggccctccctgtcactgccctgcttctcccctcgcactcctgctccacgccgctagaccacccggatggtttctggactctctgtgtgactgagggcgataatgcgaccttcacgtgctcgtctctccaa cacctccgaatcattcgtgctgaactggtaccgcatgagccccgtcaaaccagaccgaccgaccgacaa cacctccgaatcattcgtgctgaactggtaccgcatgagccc
  • the agent which enhances the activity of a CAR-expressing cell can be a costimulatory molecule or costimulatory molecule ligand.
  • costimulatory molecules include MHC class I molecule, BTLA and a Toll ligand receptor, as well as 0X40, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla/CD18), ICOS (CD278), and 4-1BB (CD137).
  • costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, IT GAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM,
  • costimulatory molecule ligands examples include CD80, CD86, CD40L, ICOSL, CD70, OX40L, 4-1BBL, GITRL, and LIGHT.
  • the costimulatory molecule ligand is a ligand for a costimulatory molecule different from the costimulatory molecule domain of the CAR.
  • the costimulatory molecule ligand is a ligand for a costimulatory molecule that is the same as the costimulatory molecule domain of the CAR.
  • the costimulatory molecule ligand is 4-1BBL.
  • the costimulatory ligand is CD80 or CD86.
  • the costimulatory molecule ligand is CD70.
  • a CAR-expressing immune effector cell described herein can be further engineered to express one or more additional costimulatory molecules or costimulatory molecule ligands.
  • the CAR-expressing cell described herein, e.g., CD 19 CAR-expressing cell further comprises a chemokine receptor molecule.
  • Transgenic expression of chemokine receptors CCR2b or CXCR2 in T cells enhances trafficking to CCL2- or CXCL1- secreting solid tumors including melanoma and neuroblastoma (Craddock et ah, J Immunother. 2010 Oct; 33(8):780-8 and Kershaw et ah, Hum Gene Ther. 2002 Nov 1; 13(16): 1971-80).
  • chemokine receptors expressed in CAR-expressing cells that recognize chemokines secreted by tumors, e.g., solid tumors, can improve homing of the CAR-expressing cell to the tumor, facilitate the infiltration of the CAR- expressing cell to the tumor, and enhances antitumor efficacy of the CAR-expressing cell.
  • the chemokine receptor molecule can comprise a naturally occurring or recombinant chemokine receptor or a chemokine-binding fragment thereof.
  • a chemokine receptor molecule suitable for expression in a CAR-expressing cell include a CXC chemokine receptor (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7), a CC chemokine receptor (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), a CX3C chemokine receptor (e.g., CX3CR1), a XC chemokine receptor (e.g., XCR1), or a chemokine-binding fragment thereof.
  • a CXC chemokine receptor e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7
  • CC chemokine receptor e.g., CCR1, CCR2, CCR3, CCR4, CCR5, C
  • the chemokine receptor molecule to be expressed with a CAR described herein is selected based on the chemokine(s) secreted by the tumor.
  • the CAR-expressing cell described herein further comprises, e.g., expresses, a CCR2b receptor or a CXCR2 receptor.
  • the CAR described herein and the chemokine receptor molecule are on the same vector or are on two different vectors. In embodiments where the CAR described herein and the chemokine receptor molecule are on the same vector, the CAR and the chemokine receptor molecule are each under control of two different promoters or are under the control of the same promoter.
  • the present invention also provides an immune effector cell, e.g., made by a method described herein, that includes a nucleic acid molecules encoding one or more CAR constructs described herein.
  • the nucleic acid molecule is provided as a messenger RNA transcript.
  • the nucleic acid molecule is provided as a DNA construct.
  • the nucleic acid molecules described herein can be a DNA molecule, an RNA molecule, or a combination thereof.
  • the nucleic acid molecule is an mRNA encoding a CAR polypeptide as described herein.
  • the nucleic acid molecule is a vector that includes any of the aforesaid nucleic acid molecules.
  • the antigen binding domain of a CAR of the invention is encoded by a nucleic acid molecule whose sequence has been codon optimized for expression in a mammalian cell.
  • entire CAR construct of the invention is encoded by a nucleic acid molecule whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences.
  • a variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least US Patent Numbers 5,786,464 and 6,114,148.
  • an immune effector cell e.g., made by a method described herein, includes a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to a tumor antigen described herein, a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) comprising a stimulatory domain, e.g., a costimulatory signaling domain (e.g., a costimulatory signaling domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein, e.g., a zeta chain described herein).
  • CAR chimeric antigen receptor
  • the present invention also provides vectors in which a nucleic acid molecule encoding a CAR, e.g., a nucleic acid molecule described herein, is inserted.
  • Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • a retroviral vector may also be, e.g., a gammaretroviral vector.
  • a gammaretroviral vector may include, e.g., a promoter, a packaging signal (y), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and a transgene of interest, e.g., a gene encoding a CAR.
  • a gammaretroviral vector may lack viral structural gens such as gag, pol, and env.
  • Exemplary gammaretroviral vectors include Murine Leukemia Vims (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom.
  • gammaretroviral vectors are described, e.g., in Tobias Maetzig et ah, “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 Jun; 3(6): 677-713.
  • the vector comprising the nucleic acid encoding the desired CAR is an adenoviral vector (A5/35).
  • the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology 9.10: 704-716, is incorporated herein by reference.
  • the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector.
  • the vectors can be suitable for replication and integration eukaryotes.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal vims, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals.
  • Vimses, which are useful as vectors include, but are not limited to, retrovimses, adenovimses, adeno- associated vimses, herpes vimses, and lentivimses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • a number of viral based systems have been developed for gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant vims can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • promoter elements regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • Exemplary promoters include the CMV IE gene, EF-la, ubiquitin C, or phosphoglycerokinase (PGK) promoters.
  • the native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from nucleic acid molecules cloned into a lentiviral vector. See, e.g., Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009).
  • the EFla promoter comprises the sequence provided in the Examples.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor vims (MMTV), human immunodeficiency vims (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor vims
  • HSV human immunodefic
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • a promoter is the phosphogly cerate kinase (PGK) promoter.
  • PGK phosphogly cerate kinase
  • a truncated PGK promoter e.g ., a PGK promoter with one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400, nucleotide deletions when compared to the wild-type PGK promoter sequence
  • PGK phosphogly cerate kinase
  • nucleotide sequences of exemplary PGK promoters are provided below.
  • PGK100 ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACG CGAGGCCTCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGA TGGCGGGGTG (SEQ ID NO: 110)
  • a vector may also include, e.g. , a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator (e.g., from Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g. SV40 origin and ColEl or others known in the art) and/or elements to allow selection (e.g . , ampicillin resistance gene and/or zeocin marker).
  • BGH Bovine Growth Hormone
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic -resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et ah, 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.
  • the vector may comprise two or more nucleic acid sequences encoding a CAR, e.g., a CAR described herein, e.g., a CD 19 CAR, and a second CAR, e.g., an inhibitory CAR or a CAR that specifically binds to an antigen other than CD 19.
  • a CAR e.g., a CAR described herein, e.g., a CD 19 CAR
  • a second CAR e.g., an inhibitory CAR or a CAR that specifically binds to an antigen other than CD 19.
  • the two or more nucleic acid sequences encoding the CAR are encoded by a single nucleic molecule in the same frame and as a single polypeptide chain.
  • the two or more CARs can, e.g., be separated by one or more peptide cleavage sites (e.g., an auto-cleavage site or a substrate for an intracellular protease).
  • peptide cleavage sites include T2A, P2A, E2A, or F2A sites.
  • Methods of introducing and expressing genes into a cell are known in the art.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method, e.g., one known in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et ah, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY). A suitable method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.
  • the CAR molecule described herein comprises one or more components of a natural killer cell receptor (NKR), thereby forming an NKR-CAR.
  • the NKR component can be a transmembrane domain, a hinge domain, or a cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptor (KIR), e.g., KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1; natural cytotoxicity receptor (NCR), e.g., NKp30, NKp44, NKp46; signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-
  • NKR-CAR molecules described herein may interact with an adaptor molecule or intracellular signaling domain, e.g., DAP12.
  • an adaptor molecule or intracellular signaling domain e.g., DAP12.
  • DAP12 intracellular signaling domain
  • Exemplary configurations and sequences of CAR molecules comprising NKR components are described in International Publication No. WO2014/145252, the contents of which are hereby incorporated by reference.
  • the CAR-expressing cell uses a split CAR.
  • the split CAR approach is described in more detail in publications WO2014/055442 and WO2014/055657.
  • a split CAR system comprises a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 4 IBB), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g., CD3 zeta).
  • the costimulatory domain is activated, and the cell proliferates.
  • the intracellular signaling domain is activated and cell-killing activity begins.
  • the CAR-expressing cell is only fully activated in the presence of both antigens.
  • a regulatable CAR where the CAR activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy.
  • CAR activities can be regulated. For example, inducible apoptosis using, e.g., a caspase fused to a dimerization domain (see, e.g., Di Stasa et al., N Engl. J. Med. 2011 Nov. 3;
  • the cells e.g., T cells or NK cells
  • a CAR of the present invention further comprise an inducible apoptosis switch, wherein a human caspase (e.g., caspase 9) or a modified version is fused to a modification of the human FKB protein that allows conditional dimerization.
  • a human caspase e.g., caspase 9
  • a modified version is fused to a modification of the human FKB protein that allows conditional dimerization.
  • the inducible caspase (e.g., caspase 9) is activated and leads to the rapid apoptosis and death of the cells (e.g., T cells or NK cells) expressing a CAR of the present invention.
  • caspase-based inducible apoptosis switch (or one or more aspects of such a switch) have been described in, e.g., US2004040047; US20110286980; US20140255360; WO1997031899; W02014151960; WO2014164348; WO2014197638; WO2014197638; all of which are incorporated by reference herein.
  • CAR-expressing cells can also express an inducible Caspase-9 (iCaspase-9) molecule that, upon administration of a dimerizer drug (e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)) leads to activation of the Caspase-9 and apoptosis of the cells.
  • a dimerizer drug e.g., rimiducid (also called AP1903 (Bellicum Pharmaceuticals) or AP20187 (Ariad)
  • AP1903 also called AP1903 (Bellicum Pharmaceuticals)
  • AP20187 AP20187
  • the iCaspase-9 molecule is encoded by a nucleic acid molecule separate from the CAR-encoding vector(s). In some cases, the iCaspase-9 molecule is encoded by the same nucleic acid molecule as the CAR-encoding vector.
  • the iCaspase-9 can provide a safety switch to avoid any toxicity of CAR-expressing cells. See, e.g., Song et al. Cancer Gene Ther. 2008; 15(10):667-75; Clinical Trial Id. No. NCT02107963; and Di Stasi et al. N. Engl. J. Med. 2011; 365:1673-83.
  • Alternative strategies for regulating the CAR therapy of the instant invention include utilizing small molecules or antibodies that deactivate or turn off CAR activity, e.g., by deleting CAR-expressing cells, e.g., by inducing antibody dependent cell-mediated cytotoxicity (ADCC).
  • CAR-expressing cells described herein may also express an antigen that is recognized by molecules capable of inducing cell death, e.g., ADCC or complement- induced cell death.
  • CAR expressing cells described herein may also express a receptor capable of being targeted by an antibody or antibody fragment.
  • receptors examples include EpCAM, VEGFR, integrins (e.g., integrins anb3, a4, aI3 ⁇ 4b3, a4b7, a5b1, anb3, an), members of the TNF receptor superfamily (e.g., TRAIL-R1 , TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1 , HLA-DR, CEA, CA-125, MUC1 , TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD1 1 , CD1 1 a/LFA-1 , CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41 , CD44, CD51 , CD52, CD62L, CD74, CD80, CD125, CD147/basigin, CD152/CT
  • a CAR-expressing cell described herein may also express a truncated epidermal growth factor receptor (EGFR) which lacks signaling capacity but retains the epitope that is recognized by molecules capable of inducing ADCC, e.g., cetuximab (ERBITUX®), such that administration of cetuximab induces ADCC and subsequent depletion of the CAR- expressing cells (see, e.g., WO2011/056894, and Jonnalagadda et ah, Gene Ther. 2013; 20(8)853-860).
  • EGFR epidermal growth factor receptor
  • Another strategy includes expressing a highly compact marker/suicide gene that combines target epitopes from both CD32 and CD20 antigens in the CAR-expressing cells described herein, which binds rituximab, resulting in selective depletion of the CAR-expressing cells, e.g., by ADCC (see, e.g., Philip et ah, Blood. 2014; 124(8)1277-1287).
  • Other methods for depleting CAR-expressing cells described herein include administration of CAMPATH, a monoclonal anti-CD52 antibody that selectively binds and targets mature lymphocytes, e.g., CAR-expressing cells, for destruction, e.g., by inducing ADCC.
  • the CAR-expressing cell can be selectively targeted using a CAR ligand, e.g., an anti-idiotypic antibody.
  • the anti-idiotypic antibody can cause effector cell activity, e.g., ADCC or ADC activities, thereby reducing the number of CAR-expressing cells.
  • the CAR ligand, e.g., the anti-idiotypic antibody can be coupled to an agent that induces cell killing, e.g., a toxin, thereby reducing the number of CAR-expressing cells.
  • the CAR molecules themselves can be configured such that the activity can be regulated, e.g., turned on and off, as described below.
  • a CAR-expressing cell described herein may also express a target protein recognized by the T cell depleting agent.
  • the target protein is CD20 and the T cell depleting agent is an anti-CD20 antibody, e.g., rituximab.
  • the T cell depleting agent is administered once it is desirable to reduce or eliminate the CAR-expressing cell, e.g., to mitigate the CAR induced toxicity.
  • the T cell depleting agent is an anti-CD52 antibody, e.g., alemtuzumab, as described in the Examples herein.
  • an RCAR comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR described herein, e.g., an antigen binding domain and an intracellular signalling domain, are partitioned on separate polypeptides or members.
  • the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signalling domain.
  • a CAR of the present invention utilizes a dimerization switch as those described in, e.g., WO2014127261, which is incorporated by reference herein.
  • an RCAR involves a switch domain, e.g., a FKBP switch domain, as set out SEQ ID NO: 114, or comprise a fragment of FKBP having the ability to bind with FRB, e.g., as set out in SEQ ID NO: 115.
  • the RCAR involves a switch domain comprising a FRB sequence, e.g., as set out in SEQ ID NO: 116, or a mutant FRB sequence, e.g., as set out in any of SEQ ID Nos. 117-122 in Table 13A.
  • Table 13A Exemplary mutant FRB having increased affinity for a dimerization molecule.
  • RNA CAR Disclosed herein are methods for producing an in vitro transcribed RNA CAR. RNA CAR and methods of using the same are described, e.g., in paragraphs 553-570 of in
  • An immune effector cell can include a CAR encoded by a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA encoding a CAR described herein is introduced into an immune effector cell, e.g., made by a method described herein, for production of a CAR-expressing cell.
  • the in vitro transcribed RNA CAR can be introduced to a cell as a form of transient transfection.
  • the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
  • DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired temple for in vitro transcription is a CAR described herein.
  • the template for the RNA CAR comprises an extracellular region comprising a single chain variable domain of an antibody to a tumor associated antigen described herein; a hinge region ( e.g ., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein such as a transmembrane domain of CD8a); and a cytoplasmic region that includes an intracellular signaling domain, e.g., an intracellular signaling domain described herein, e.g., comprising the signaling domain of CD3-zeta and the signaling domain of 4- IBB.
  • an intracellular signaling domain e.g., an intracellular signaling domain described herein, e.g., comprising the signaling domain of CD3-zeta and the signaling domain of 4- IBB.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the nucleic acid can include some or all of the 5' and/or 3' untranslated regions (UTRs).
  • the nucleic acid can include exons and introns.
  • the DNA to be used for PCR is a human nucleic acid sequence.
  • the DNA to be used for PCR is a human nucleic acid sequence including the 5' and 3' UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection.
  • Methods for performing PCR are well known in the art.
  • Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
  • “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
  • the primers can be designed to be substantially complementary to any portion of the DNA template.
  • the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs.
  • the primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest.
  • the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs.
  • Primers useful for PCR can be generated by synthetic methods that are well known in the art.
  • “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
  • Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
  • reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
  • Downstream is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand.
  • DNA polymerase useful for PCR can be used in the methods disclosed herein.
  • the reagents and polymerase are commercially available from a number of sources.
  • the RNA in embodiments has 5' and 3' UTRs.
  • the 5' UTR is between one and 3000 nucleotides in length.
  • the length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the nucleic acid of interest.
  • UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5' UTR can contain the Kozak sequence of the endogenous nucleic acid.
  • a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art.
  • the 5' UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (SEQ ID NO: 123) (size can be 50-5000 T (SEQ ID NO: 32)), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA.
  • the poly(A) tail is between 100 and 5000 adenosines ( e.g ., SEQ ID NO: 33).
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP).
  • E-PAP E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides (SEQ ID NO: 34) results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods disclosed herein include a 5' cap.
  • the 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).
  • RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
  • Non-viral delivery methods include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instrument
  • non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject.
  • the non-viral method includes the use of a transposon (also called a transposable element).
  • a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome.
  • a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.
  • Exemplary methods of nucleic acid delivery using a transposon include a Sleeping Beauty transposon system (SBTS) and a piggyBac (PB) transposon system.
  • SBTS Sleeping Beauty transposon system
  • PB piggyBac
  • the SBTS includes two components: 1) a transposon containing a transgene and 2) a source of transposase enzyme.
  • the transposase can transpose the transposon from a carrier plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome/genome.
  • a target DNA such as a host cell chromosome/genome.
  • the transposase binds to the carrier plasmid/donor DNA, cuts the transposon (including transgene(s)) out of the plasmid, and inserts it into the genome of the host cell. See, e.g., Aronovich et al. supra.
  • Exemplary transposons include a pT2-based transposon. See, e.g., Grabundzija et al. Nucleic Acids Res. 41.3(2013): 1829-47; and Singh et al. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporated herein by reference.
  • Exemplary transposases include a Tel /mariner- type transposase, e.g., the SB 10 transposase or the SB 11 transposase (a hyperactive transposase which can be expressed, e.g., from a cytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.; and Grabundzija et al., all of which are incorporated herein by reference.
  • SBTS permits efficient integration and expression of a transgene, e.g. , a nucleic acid encoding a CAR described herein.
  • a transgene e.g. , a nucleic acid encoding a CAR described herein.
  • one or more nucleic acids e.g., plasmids, containing the SBTS components are delivered to a cell (e.g., T or NK cell).
  • the nucleic acid(s) are delivered by standard methods of nucleic acid (e.g., plasmid DNA) delivery, e.g., methods described herein, e.g., electroporation, transfection, or lipofection.
  • the nucleic acid contains a transposon comprising a transgene, e.g., a nucleic acid encoding a CAR described herein.
  • the nucleic acid contains a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR described herein) as well as a nucleic acid sequence encoding a transposase enzyme.
  • a system with two nucleic acids is provided, e.g., a dual-plasmid system, e.g., where a first plasmid contains a transposon comprising a transgene, and a second plasmid contains a nucleic acid sequence encoding a transposase enzyme.
  • the first and the second nucleic acids are co-delivered into a host cell.
  • cells e.g., T or NK cells
  • a CAR described herein by using a combination of gene insertion using the SBTS and genetic editing using a nuclease (e.g., Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system, or engineered meganuclease re-engineered homing endonucleases).
  • ZFNs Zinc finger nucleases
  • TALENs Transcription Activator-Like Effector Nucleases
  • CRISPR/Cas system or engineered meganuclease re-engineered homing endonucleases
  • use of a non-viral method of delivery permits reprogramming of cells, e.g., T or NK cells, and direct infusion of the cells into a subject.
  • Advantages of non-viral vectors include but are not limited to the ease and relatively low cost of producing sufficient amounts required to meet a patient population, stability during storage, and lack of immunogenicity .
  • the methods disclosed herein further include administering a T cell depleting agent after treatment with the cell (e.g., an immune effector cell as described herein), thereby reducing (e.g., depleting) the CAR-expressing cells (e.g., the CD19CAR- expressing cells).
  • a T cell depleting agent after treatment with the cell (e.g., an immune effector cell as described herein), thereby reducing (e.g., depleting) the CAR-expressing cells (e.g., the CD19CAR- expressing cells).
  • T cell depleting agents can be used to effectively deplete CAR- expressing cells (e.g ., CD19CAR-expressing cells) to mitigate toxicity.
  • the CAR-expressing cells were manufactured according to a method herein, e.g., assayed (e.g., before or after transfection or transduction) according to a method herein.
  • the T cell depleting agent is administered one, two, three, four, or five weeks after administration of the cell, e.g., the population of immune effector cells, described herein.
  • the T cell depleting agent is an agent that depletes CAR-expressing cells, e.g., by inducing antibody dependent cell-mediated cytotoxicity (ADCC) and/or complement-induced cell death.
  • CAR-expressing cells described herein may also express an antigen (e.g., a target antigen) that is recognized by molecules capable of inducing cell death, e.g., ADCC or complement- induced cell death.
  • CAR expressing cells described herein may also express a target protein (e.g., a receptor) capable of being targeted by an antibody or antibody fragment.
  • target proteins include, but are not limited to, EpCAM, VEGFR, integrins (e.g., integrins anb3, a4, aI3/4b3, a4b7, a5b1, anb3, an), members of the TNF receptor superfamily (e.g., TRAIL-R1 , TRAIL-R2), PDGF Receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11 , CDlla/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/lgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41 , CD44, CD51 , CD52, CD62L, CD74, CD80, CD 125, CD147/basigin, CD152/CT
  • the CAR expressing cell co-expresses the CAR and the target protein, e.g., naturally expresses the target protein or is engineered to express the target protein.
  • the cell e.g., the population of immune effector cells, can include a nucleic acid (e.g., vector) comprising the CAR nucleic acid (e.g., a CAR nucleic acid as described herein) and a nucleic acid encoding the target protein.
  • the T cell depleting agent is a CD52 inhibitor, e.g., an anti- CD52 antibody molecule, e.g., alemtuzumab.
  • the cell e.g., the population of immune effector cells, expresses a CAR molecule as described herein (e.g., CD19CAR) and the target protein recognized by the T cell depleting agent.
  • the target protein is CD20.
  • the T cell depleting agent is an anti-CD20 antibody, e.g., rituximab.
  • the methods further include transplanting a cell, e.g., a hematopoietic stem cell, or a bone marrow, into the mammal.
  • a cell e.g., a hematopoietic stem cell, or a bone marrow
  • the invention features a method of conditioning a mammal prior to cell transplantation.
  • the method includes administering to the mammal an effective amount of the cell comprising a CAR nucleic acid or polypeptide, e.g., a CD 19 CAR nucleic acid or polypeptide.
  • the cell transplantation is a stem cell transplantation, e.g., a hematopoietic stem cell transplantation, or a bone marrow transplantation.
  • conditioning a subject prior to cell transplantation includes reducing the number of target-expressing cells in a subject, e.g., CD 19-expressing normal cells or CD 19-expressing cancer cells.
  • Immune effector cells such as T cells generated or enriched by the methods described herein may be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.
  • a population of immune effector cells may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody can be used.
  • an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Bcsancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med.
  • the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols.
  • the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation).
  • one agent may be coupled to a surface and the other agent in solution.
  • the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain aspects, both agents can be in solution.
  • the agents may be in soluble form, and then cross- linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • a surface such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents.
  • aAPCs artificial antigen presenting cells
  • the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.”
  • the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co -immobilized to the same bead in equivalent molecular amounts.
  • a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used.
  • a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1:1. In one particular aspect an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1:1.
  • the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between. In one aspect, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain aspects, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1.
  • a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further aspect, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.
  • Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T cells or other target cells.
  • the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many.
  • the ratio of cells to particles ranges from 1:100 to 100:1 and any integer values in-between and in further aspects the ratio comprises 1:9 to 9:1 and any integer values in between, can also be used to stimulate T cells.
  • the ratio of anti-CD3- and anti-CD28- coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain suitable values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4,
  • a ratio of particles to cells of 1:1 or less is used.
  • a suitable particle: cell ratio is 1:5.
  • the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is from 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (based on cell counts on the day of addition).
  • the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:5 on the third and fifth days of stimulation. In one aspect, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation.
  • ratios will vary depending on particle size and on cell size and type. In one aspect, the most typical ratios for use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day.
  • the cells such as T cells
  • the cells are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.
  • cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells.
  • the cells for example, 10 4 to 10 9 T cells
  • beads for example, DYNABEADS® M- 450 CD3/CD28 T paramagnetic beads at a ratio of 1:1
  • a buffer for example PBS (without divalent cations such as, calcium and magnesium).
  • the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest.
  • any cell number is within the context of the present invention.
  • it may be desirable to significantly decrease the volume in which particles and cells are mixed together i.e., increase the concentration of cells, to ensure maximum contact of cells and particles.
  • a concentration of about 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, 5 billion/ml, or 2 billion cells/ml is used.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further aspects, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain aspects. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • cells transduced with a nucleic acid encoding a CAR are expanded, e.g., by a method described herein.
  • the cells are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days).
  • the cells are expanded for a period of 4 to 9 days.
  • the cells are expanded for a period of 8 days or less, e.g., 7, 6 or 5 days.
  • the cells are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, e.g., by various T cell functions, e.g. proliferation, target cell killing, cytokine production, activation, migration, or combinations thereof.
  • the cells e.g., a CD19 CAR cell described herein, expanded for 5 days show at least a one, two, three or four fold increase in cells doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions.
  • the cells e.g., the cells expressing a CD19 CAR described herein, are expanded in culture for 5 days, and the resulting cells exhibit higher proinflammatory cytokine production, e.g., IFN-g and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions.
  • proinflammatory cytokine production e.g., IFN-g and/or GM-CSF levels
  • the cells e.g., a CD 19 CAR cell described herein, expanded for 5 days show at least a one, two, three, four, five, ten fold or more increase in pg/ml of proinflammatory cytokine production, e.g., IFN-g and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions.
  • proinflammatory cytokine production e.g., IFN-g and/or GM-CSF levels
  • T cell culture includes an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives for the growth of cells known to the skilled artisan.
  • serum e.g., fetal bovine or human serum
  • IL-2 interleukin-2
  • insulin IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives for the growth of cells known to the skilled artisan.
  • additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.
  • Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum- free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells.
  • Antibiotics e.g., penicillin and streptomycin
  • the target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5%
  • the cells are expanded in an appropriate media (e.g., media described herein) that includes one or more interleukin that result in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in cells over a 14 day expansion period, e.g., as measured by a method described herein such as flow cytometry.
  • the cells are expanded in the presence IL-15 and/or IL-7 (e.g., IL-15 and IL-7).
  • methods described herein comprise removing T regulatory cells, e.g., CD25+ T cells, from a cell population, e.g., using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2.
  • T regulatory cells e.g., CD25+ T cells
  • methods of removing T regulatory cells, e.g., CD25+ T cells, from a cell population are described herein.
  • the methods further comprise contacting a cell population (e.g., a cell population in which T regulatory cells, such as CD25+ T cells, have been depleted; or a cell population that has previously contacted an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15 and/or IL-7.
  • a cell population e.g., a cell population in which T regulatory cells, such as CD25+ T cells, have been depleted; or a cell population that has previously contacted an anti-CD25 antibody, fragment thereof, or CD25-binding ligand
  • the cell population e.g., that has previously contacted an anti-CD25 antibody, fragment thereof, or CD25-binding ligand
  • a CAR-expressing cell described herein is contacted with a composition comprising a interleukin- 15 (IL-15) polypeptide, a interleukin- 15 receptor alpha (IL-15Ra) polypeptide, or a combination of both a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15, during the manufacturing of the CAR-expressing cell, e.g., ex vivo.
  • a CAR-expressing cell described herein is contacted with a composition comprising a IL-15 polypeptide during the manufacturing of the CAR-expressing cell, e.g., ex vivo.
  • a CAR-expressing cell described herein is contacted with a composition comprising a combination of both a IL-15 polypeptide and a IL-15 Ra polypeptide during the manufacturing of the CAR-expressing cell, e.g., ex vivo.
  • a CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during the manufacturing of the CAR-expressing cell, e.g., ex vivo.
  • the CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during ex vivo expansion.
  • the CAR- expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during ex vivo expansion.
  • the CAR-expressing cell described herein is contacted with a composition comprising both an IL-15 polypeptide and an IL-15Ra polypeptide during ex vivo expansion.
  • the contacting results in the survival and proliferation of a lymphocyte subpopulation, e.g., CD8+ T cells.
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+).
  • TH, CD4+ helper T cell population
  • TC cytotoxic or suppressor T cell population
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells.
  • infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
  • an antigen- specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • CD4 and CD8 markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.
  • a CAR described herein is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re- stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a CAR of the present invention are described in further detail below
  • Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers, e.g., as described in paragraph 695 of International Application WO2015/ 142675, filed March 13, 2015, which is herein incorporated by reference in its entirety.
  • In vitro expansion of CAR + T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4 + and CD8 + T cells are stimulated with aCD3/aCD28 aAPCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed.
  • Exemplary promoters include the CMV IE gene, EF-la, ubiquitin C, or phosphoglycerokinase (PGK) promoters.
  • GFP fluorescence is evaluated on day 6 of culture in the CD4 + and/or CD8 + T cell subsets by flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
  • a mixture of CD4 + and CD8 + T cells are stimulated with aCD3/aCD28 coated magnetic beads on day 0, and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using a 2A ribosomal skipping sequence.
  • Cultures are re-stimulated with either a cancer associated antigen as described herein + K562 cells (K562-expressing a cancer associated antigen as described herein), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1BBL in the presence of antiCD3 and anti-CD28 antibody (K562-BBL-3/28) following washing.
  • Exogenous IL-2 is added to the cultures every other day at 100 IU/ml.
  • GFP + T cells are enumerated by flow cytometry using bead-based counting. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
  • Sustained CAR + T cell expansion in the absence of re- stimulation can also be measured. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter, a Nexcelom Cellometer Vision or Millipore Scepter, following stimulation with aCD3/aCD28 coated magnetic beads on day 0, and transduction with the indicated CAR on day 1.
  • Animal models can also be used to measure a CAR-expressing cell activity, e.g., as described in paragraph 698 of International Application WO2015/142675, filed March 13, 2015, which is herein incorporated by reference in its entirety.
  • Dose dependent CAR treatment response can be evaluated, e.g., as described in paragraph 699 of International Application WO2015/ 142675, filed March 13, 2015, which is herein incorporated by reference in its entirety.
  • Cytotoxicity can also be assessed by measuring changes in adherent cell’s electrical impedance, e.g., using an xCELLigence real time cell analyzer (RTCA). In some embodiments, cytotoxicity is measured at multiple time points.
  • RTCA real time cell analyzer
  • Imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumor-bearing animal models, e.g., as described in paragraph 702 of International Application WO2015/ 142675, filed March 13, 2015, which is herein incorporated by reference in its entirety.
  • the CAR ligand is an antibody that binds to the CAR molecule, e.g., binds to the extracellular antigen binding domain of CAR (e.g., an antibody that binds to the antigen binding domain, e.g., an anti-idiotypic antibody; or an antibody that binds to a constant region of the extracellular binding domain).
  • the CAR ligand is a CAR antigen molecule (e.g., a CAR antigen molecule as described herein).
  • a method for detecting and/or quantifying CAR-expressing cells is disclosed.
  • the CAR ligand can be used to detect and/or quantify CAR-expressing cells in vitro or in vivo (e.g., clinical monitoring of CAR-expressing cells in a patient, or dosing a patient).
  • the method includes: providing the CAR ligand (optionally, a labelled CAR ligand, e.g., a CAR ligand that includes a tag, a bead, a radioactive or fluorescent label); acquiring the CAR-expressing cell (e.g., acquiring a sample containing CAR-expressing cells, such as a manufacturing sample or a clinical sample); contacting the CAR-expressing cell with the CAR ligand under conditions where binding occurs, thereby detecting the level (e.g., amount) of the CAR-expressing cells present. Binding of the CAR-expressing cell with the CAR ligand can be detected using standard techniques such as FACS, ELISA and the like.
  • a method of expanding and/or activating cells includes: providing a CAR-expressing cell (e.g., a first CAR-expressing cell or a transiently expressing CAR cell); contacting said CAR-expressing cell with a CAR ligand, e.g., a CAR ligand as described herein), under conditions where immune cell expansion and/or proliferation occurs, thereby producing the activated and/or expanded cell population.
  • a CAR-expressing cell e.g., a first CAR-expressing cell or a transiently expressing CAR cell
  • a CAR ligand e.g., a CAR ligand as described herein
  • the CAR ligand is present on a substrate (e.g., is immobilized or attached to a substrate, e.g., a non-naturally occurring substrate).
  • the substrate is a non-cellular substrate.
  • the non-cellular substrate can be a solid support chosen from, e.g., a plate (e.g., a microtiter plate), a membrane (e.g., a nitrocellulose membrane), a matrix, a chip or a bead.
  • the CAR ligand is present in the substrate (e.g., on the substrate surface).
  • the CAR ligand can be immobilized, attached, or associated covalently or non-covalently (e.g., cross-linked) to the substrate.
  • the CAR ligand is attached (e.g., covalently attached) to a bead.
  • the immune cell population can be expanded in vitro or ex vivo.
  • the method can further include culturing the population of immune cells in the presence of the ligand of the CAR molecule, e.g., using any of the methods described herein.
  • the method of expanding and/or activating the cells further comprises addition of a second stimulatory molecule, e.g., CD28.
  • a second stimulatory molecule e.g., CD28.
  • the CAR ligand and the second stimulatory molecule can be immobilized to a substrate, e.g., one or more beads, thereby providing increased cell expansion and/or activation.
  • a method for selecting or enriching for a CAR expressing cell includes contacting the CAR expressing cell with a CAR ligand as described herein; and selecting the cell on the basis of binding of the CAR ligand.
  • a method for depleting, reducing and/or killing a CAR expressing cell is provided. The method includes contacting the CAR expressing cell with a CAR ligand as described herein; and targeting the cell on the basis of binding of the CAR ligand, thereby reducing the number, and/or killing, the CAR-expressing cell.
  • the CAR ligand is coupled to a toxic agent (e.g ., a toxin or a cell ablative drug).
  • the anti-idiotypic antibody can cause effector cell activity, e.g., ADCC or ADC activities.
  • anti-CAR antibodies that can be used in the methods disclosed herein are described, e.g., in WO 2014/190273 and by Jena et ah, “Chimeric Antigen Receptor (CAR)- Specific Monoclonal Antibody to Detect CD 19-Specific T cells in Clinical Trials”, PLOS March 2013 8:3 e57838, the contents of which are incorporated by reference.
  • CAR Chimeric Antigen Receptor
  • compositions and methods herein are optimized for a specific subset of T cells, e.g., as described in US Serial No. PCT/US2015/043219 filed July 31, 2015, the contents of which are incorporated herein by reference in their entirety.
  • the optimized subsets of T cells display an enhanced persistence compared to a control T cell, e.g., a T cell of a different type (e.g., CD8+ or CD4+) expressing the same construct.
  • a CD4+ T cell comprises a CAR described herein, which CAR comprises an intracellular signaling domain suitable for (e.g., optimized for, e.g., leading to enhanced persistence in) a CD4+ T cell, e.g., an ICOS domain.
  • a CD8+ T cell comprises a CAR described herein, which CAR comprises an intracellular signaling domain suitable for (e.g., optimized for, e.g., leading to enhanced persistence of) a CD8+ T cell, e.g., a 4- IBB domain, a CD28 domain, or another costimulatory domain other than an ICOS domain.
  • the CAR described herein comprises an antigen binding domain described herein, e.g., a CAR comprising an antigen binding domain.
  • a method of treating a subject e.g., a subject having cancer.
  • the method includes administering to said subject, an effective amount of:
  • a CD4+ T cell comprising a CAR (the CARCD4+) comprising: an antigen binding domain, e.g., an antigen binding domain described herein; a transmembrane domain; and an intracellular signaling domain, e.g., a first costimulatory domain, e.g., an ICOS domain; and
  • a CD8+ T cell comprising a CAR (the CARCD8+) comprising: an antigen binding domain, e.g., an antigen binding domain described herein; a transmembrane domain; and an intracellular signaling domain, e.g., a second costimulatory domain, e.g., a 4- IBB domain, a CD28 domain, or another costimulatory domain other than an ICOS domain; wherein the CARCD4+ and the CARCD8+ differ from one another.
  • a CAR the CARCD8+
  • an antigen binding domain e.g., an antigen binding domain described herein
  • a transmembrane domain e.g., an intracellular signaling domain, e.g., a second costimulatory domain, e.g., a 4- IBB domain, a CD28 domain, or another costimulatory domain other than an ICOS domain
  • the method further includes administering:
  • a second CD8+ T cell comprising a CAR (the second CARCD8+) comprising: an antigen binding domain, e.g., an antigen binding domain described herein; a transmembrane domain; and an intracellular signaling domain, wherein the second CARCD8+ comprises an intracellular signaling domain, e.g., a costimulatory signaling domain, not present on the CARCD8+, and, optionally, does not comprise an ICOS signaling domain.
  • the second CARCD8+ comprises an intracellular signaling domain, e.g., a costimulatory signaling domain, not present on the CARCD8+, and, optionally, does not comprise an ICOS signaling domain.
  • one or more CAR-expressing cells as disclosed herein can be administered or delivered to the subject via a biopolymer scaffold, e.g., a biopolymer implant.
  • Biopolymer scaffolds can support or enhance the delivery, expansion, and/or dispersion of the CAR-expressing cells described herein.
  • a biopolymer scaffold comprises a biocompatible (e.g., does not substantially induce an inflammatory or immune response) and/or a biodegradable polymer that can be naturally occurring or synthetic. Exemplary biopolymers are described, e.g., in paragraphs 1004-1006 of International Application WO2015/142675, filed March 13, 2015, which is herein incorporated by reference in its entirety.
  • the disclosure provides a method of treating a subject, comprising administering CAR-expressing cells produced as described herein, optionally in combination with one or more other therapies. In some aspects, the disclosure provides a method of treating a subject, comprising administering a reaction mixture comprising CAR-expressing cells as described herein, optionally in combination with one or more other therapies. In some aspects, the disclosure provides a method of shipping or receiving a reaction mixture comprising CAR- expressing cells as described herein. In some aspects, the disclosure provides a method of treating a subject, comprising receiving a CAR-expressing cell that was produced as described herein, and further comprising administering the CAR-expressing cell to the subject, optionally in combination with one or more other therapies.
  • the disclosure provides a method of treating a subject, comprising producing a CAR-expressing cell as described herein, and further comprising administering the CAR-expressing cell to the subject, optionally in combination with one or more other therapies.
  • the other therapy may be, e.g., a cancer therapy such as chemotherapy.
  • cells expressing a CAR described herein are administered to a subject in combination with a molecule that decreases the Treg cell population.
  • Methods that decrease the number of (e.g., deplete) Treg cells are known in the art and include, e.g., CD25 depletion, cyclophosphamide administration, modulating GITR function.
  • reducing the number of Treg cells in a subject prior to apheresis or prior to administration of a CAR-expressing cell described herein reduces the number of unwanted immune cells (e.g., Tregs) in the tumor microenvironment and reduces the subject’s risk of relapse.
  • a therapy described herein e.g., a CAR-expressing cell
  • a molecule targeting GITR and/or modulating GITR functions such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Tregs).
  • cells expressing a CAR described herein are administered to a subject in combination with cyclophosphamide.
  • the GITR binding molecules and/or molecules modulating GITR functions are administered prior to the CAR-expressing cell.
  • a GITR agonist can be administered prior to apheresis of the cells.
  • cyclophosphamide is administered to the subject prior to administration (e.g., infusion or re-infusion) of the CAR-expressing cell or prior to apheresis of the cells.
  • cyclophosphamide and an anti-GITR antibody are administered to the subject prior to administration (e.g., infusion or re-infusion) of the CAR-expressing cell or prior to apheresis of the cells.
  • the subject has cancer (e.g., a solid cancer or a hematological cancer such as ALL or CLL). In one embodiment, the subject has CLL.
  • the subject has ALL.
  • the subject has a solid cancer, e.g., a solid cancer described herein.
  • Exemplary GITR agonists include, e.g., GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, e.g., a GITR fusion protein described in U.S. Patent No.: 6,111,090, European Patent No.: 090505B1, U.S Patent No.: 8,586,023, PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g., in U.S.
  • a CAR expressing cell described herein is administered to a subject in combination with a GITR agonist, e.g., a GITR agonist described herein.
  • a GITR agonist e.g., a GITR agonist described herein.
  • the GITR agonist is administered prior to the CAR-expressing cell.
  • the GITR agonist can be administered prior to apheresis of the cells.
  • the subject has CLL.
  • compositions may comprise a CAR-expressing cell, e.g., a plurality of CAR-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • Compositions can be formulated, e.g., for intravenous administration.
  • the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
  • an immunologically effective amount When “an immunologically effective amount,” “an anti-cancer effective amount,” “a cancer-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune effector cells (e.g ., T cells, NK cells) described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et ah, New Eng. J. of Med. 319:1676, 1988).
  • a dose of CAR cells (e.g., CD 19 CAR cells) comprises about 1 x
  • a dose of CAR cells comprises at least about 1 x 10 6 , 1.1 x 10 6 , 2 x 10 6 , 3.6 x 10 6 , 5 x 10 6 , 1 x 10 7 , 1.8 x 10 7 , 2 x 10 7 , 5 x 10 7 , 1 x 10 8 , 2 x 10 8 , or 5 x 10 8 cells/kg.
  • a dose of CAR cells comprises up to about 1 x 10 6 , 1.1 x 10 6 , 2 x 10 6 , 3.6 x 10 6 , 5 x 10 6 , 1 x 10 7 , 1.8 x
  • a dose of CAR cells comprises about 1.1 x 10 6 - 1.8 x 10 7 cells/kg.
  • a dose of CAR cells comprises about 1 x 10 7 , 2 x 10 7 , 5 x 10 7 , 1 x 10 8 , 2 x 10 8 , 5 x 10 8 , 1 x 10 9 , 2 x 10 9 , or 5 x 10 9 cells.
  • a dose of CAR cells comprises at least about 1 x 10 7 , 2 x 10 7 , 5 x 10 7 , 1 x 10 8 , 2 x 10 8 , 5 x 10 8 , 1 x 10 9 , 2 x 10 9 , or 5 x 10 9 cells.
  • a dose of CAR cells comprises up to about 1 x 10 7 , 2 x 10 7 , 5 x 10 7 , 1 x 10 8 , 2 x 10 8 , 5 x
  • activated immune effector cells e.g ., T cells, NK cells
  • activate immune effector cells e.g., T cells, NK cells
  • reinfuse the subject with these activated and expanded immune effector cells e.g., T cells, NK cells.
  • This process can be carried out multiple times every few weeks.
  • immune effector cells e.g., T cells, NK cells
  • immune effector cells e.g., T cells, NK cells
  • T cells e.g., T cells, NK cells
  • compositions described herein may be administered to a subject trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally, e.g., by intradermal or subcutaneous injection.
  • the compositions of immune effector cells e.g., T cells, NK cells
  • T cells, NK cells may be injected directly into a tumor, lymph node, or site of infection.
  • the disclosure provides a method of treating a subject having, or at risk of having a lymphoma (e.g., a lymphoma disclosed herein, e.g. DLBCL or FL), comprising responsive to a determination, e.g., prediction, of a lesion-level treatment response to a therapy comprising a Chimeric Antigen Receptor 19 (CAR19) immune effector cell (“CAR19 therapy”), administering the CAR 19 therapy to the subject, thereby treating the subject.
  • a lymphoma e.g., a lymphoma disclosed herein, e.g. DLBCL or FL
  • a method of treating a subject having a lymphoma comprising responsive to a determination, e.g., prediction, of a lesion-level treatment response to a therapy comprising a Chimeric Antigen Receptor 19 (CAR19) immune effector cell (“CAR19 therapy”), administering the CAR 19 therapy to the subject, thereby treating the subject.
  • the disclosure provides a method of evaluating a subject having, or at risk of having a lymphoma (e.g., a lymphoma disclosed herein,), comprising determining, e.g., predicting, of a lesion-level treatment response to a therapy comprising a Chimeric Antigen Receptor 19 (CAR19) immune effector cell (“CAR19 therapy”), with a neural network.
  • determining comprises: acquiring, e.g., receiving, an image of a lesion of the subject (“acquired image”); and/or processing the image with the neural network (“processed image”).
  • the neural network outputs a classification result indicating the lesion-level treatment response to the CAR 19 therapy.
  • the subject has or has been identified as having a lymphoma, e.g., a relapsed and/or refractory lymphoma.
  • the lymphoma is chosen from: DLBCL, follicular lymphoma (FL), mantle cell lymphoma (MCL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, or plasmablastic lymphoma.
  • the lymphoma is chosen from: DLBCL,
  • relapse refers to reappearance of a disease (e.g., cancer) after an initial period of responsiveness, e.g., after prior treatment with a therapy, e.g., cancer therapy (e.g., complete response or partial response).
  • the initial period of responsiveness may involve the level of cancer cells falling below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the reappearance may involve the level of cancer cells rising above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • Refractory refers to a disease, e.g., cancer, that does not respond to a treatment.
  • a refractory cancer can be resistant to a treatment before or at the beginning of the treatment.
  • the refractory cancer can become resistant during a treatment.
  • a refractory cancer is also called a resistant cancer.
  • the subject has DLBCL. In some embodiments the subject has relapsed or refractory DLBCL. In some embodiments, the subject is at least 18 years of age.
  • the subject having DLBCL e.g., relapsed or refractory DLBCL has previously been administered one or more of: an anti-CD20 therapy, an anthracycline based chemotherapy or stem cell therapy, e.g., allogeneic or autologous SCT, e.g., as described herein, as a, e.g., first, second or third line therapy.
  • an anti-CD20 therapy e.g., an anthracycline based chemotherapy or stem cell therapy, e.g., allogeneic or autologous SCT, e.g., as described herein, as a, e.g., first, second or third line therapy.
  • the subject has no response to, e.g., relapsed, refractory, has progressive disease, or has failed, the first, second or third line therapy.
  • a subject having relapsed or refractory DLBCL is administered a combination therapy comprising a BTK inhibitor, e.g., ibmtinib, and a CAR-expressing cell, e.g., according to a dosage regimen described herein.
  • a BTK inhibitor e.g., ibmtinib
  • a CAR-expressing cell e.g., according to a dosage regimen described herein.
  • the subject has previously been treated with a BTK inhibitor, e.g., for at least 4-6 weeks or 8-10 weeks.
  • the subject is administered the BTK inhibitor, e.g., daily, prior to apheresis, e.g., at least about 21 days, e.g., 21-30 days, e.g., 28 days prior to apheresis.
  • the subject is administered the BTK inhibitor for at least about 21 days, e.g., 10- 100 days, after apheresis and prior to CAR therapy administration, e.g., infusion.
  • the subject is administered the BTK inhibitor concurrently with or after apheresis. In some embodiments, the subject is administered the BTK inhibitor for at least about 21 days, e.g., 10-100 days, after apheresis and prior to CAR therapy administration, e.g., infusion. In some embodiments, the subject is continuously administered with a BTK inhibitor, e.g., at a dose of 560mg, daily. In some embodiments, the subject is administered 0.6- 6.0 x 10 8 CAR expressing cells.
  • the subject is administered lymphodepletion after initiation of the BTK inhibitor, but prior to administration of the CAR therapy.
  • the lymphodepletion comprises administering cyclophosphamide and fludarabine.
  • the lymphodepletion comprises administering 500 mg/m2 cyclophosphamide daily for 2 days and 30 mg/m2 fludarabine daily for 3 days.
  • the lymphodepletion comprises administering 250 mg/m2 cyclophosphamide daily for 3 days, and 25 mg/m2 fludarabine daily for 3 days.
  • the lymphodepletion begins with the administration of the first dose of fludarabine.
  • cyclophosphamide and fludarabine are administered on the same day. In some embodiments, cyclophosphamide and fludarabine are not administered on the same day. In some embodiments, the daily dosages are administered on consecutive days. In embodiments, the lymphodepletion comprises administering bendamustine. In some embodiments, bendamustine is administered daily, e.g., twice daily, at a dosage of about 75-125 mg/m2 (e.g., 75-100 or 100-125 mg/m 2 , e.g., about 90 mg/m 2 ), e.g., intravenously.
  • bendamustine is administered daily, e.g., twice daily, at a dosage of about 75-125 mg/m2 (e.g., 75-100 or 100-125 mg/m 2 , e.g., about 90 mg/m 2 ), e.g., intravenously.
  • bendamustine is administered at dosage of 90 mg/m 2 daily, e.g., for 2 days.
  • the subject has a cancer, e.g., a hematological cancer as described herein.
  • the subject is administered a first lymphodepletion regimen and/or a second lymphodepletion regimen.
  • the first lymphodepletion regimen is administered before the second lymphodepletion regimen.
  • the second lymphodepletion regimen is administered before the first lymphodepletion regimen.
  • the first lymphodepletion regimen comprises cyclophosphamide and fludarabine, e.g., 250 mg/m2 cyclophosphamide daily for 3 days, and 25 mg/m2 fludarabine daily for 3 days.
  • the second lymphodepletion regimen comprises bendamustine, e.g. ,90 mg/m 2 daily, e.g., for 2 days.
  • the second lymphodepletion regimen is administered as an alternate lymphodepletion regimen, e.g., if a subject has experienced adverse effects, e.g., Grade 4 hemorrhagic cystitis, to a lymphodepletion regimen comprising cyclophosphamide.
  • the lymphoma is a DLBCL, e.g., a relapsed or refractory DLBCL (e.g., r/r DLBCL), e.g., a CD19+ r/r DLBCL.
  • the subject is an adult and the lymphoma is an r/r DLBCL.
  • a subject administered a therapy described herein e.g., a therapy comprising a CAR-expressing therapy, e.g., a therapy comprising a CAR 19-expressing therapy (e.g., a CAR 19-expressing therapy in combination with a BTK inhibitor or a PD-1 inhibitor), has previously received, e.g., been administered, one or more lines of therapy, e.g., 2, 3, 4, or 5 or more lines of therapy (e.g., one or more therapies as described herein) and/or the subject was not eligible for or had failed stem cell therapy (SCT), e.g., autologous or allogeneic SCT.
  • SCT stem cell therapy
  • the subject has previously received 2 or more lines of therapy comprising rituximab and anthracy cline. In some embodiments, the subject was not eligible for or had failed autologous SCT. In some embodiments, administration of a CAR 19-expressing therapy (e.g., in combination with a BTK inhibitor or a PD-1 inhibitor) to the subject who has previously undergone 2 or more lines of therapy and/or was not eligible for or had failed autologous SCT results in a response, e.g., a high response rate and/or a durable response to the therapy, e.g., therapy comprising a CAR 19-expressing therapy (e.g., in combination with a BTK inhibitor or a PD-1 inhibitor). In some embodiments, the subject has a hematological cancer, e.g., DLBCL, e.g., relapsed and/or refractory DLBCL.
  • DLBCL hematological cancer
  • Follicular lymphoma Follicular lymphom
  • the subject has follicular lymphoma (FL).
  • FL is also referred to as a Non-Hodgkin lymphoma.
  • the subject has relapsed or refractory FL.
  • the FL can be classified as a Stage I lymphoma, a Stage II lymphoma, a Stage III lymphoma or a Stage IV lymphoma.
  • FL and standard of care for FL is described in Luminaari S el al. (2012); Rev Bras Hematol Hemoter. 34(l):54-59, the entire contents of which is hereby incorporated by reference.
  • the subject having FL has previously been administered one or more of: a chemotherapy, immunotherapy, radiation therapy or radioimmunotherapy, e.g., as a first, second, or third line therapy.
  • a chemotherapy e.g., rituximab
  • an anti-CD20 therapy e.g., rituximab
  • an anthracy cline based chemotherapy e.g., anthracy cline based chemotherapy
  • a stem cell therapy e.g., allogeneic or autologous SCT
  • the subject has no response to, e.g., relapsed, refractory, has progressive disease, or has failed, the first, second or third line therapy.
  • Example 1 Deep-learning-based image analysis of pre-treatment diagnostic CT and PET/CT images for predicting treatment response to chimeric antigen receptor (CAR) T cell therapy in lymphoma
  • CAR chimeric antigen receptor
  • This Example describes the feasibility of applying a deep-learning (DL)-based image analysis methodology on pre-treatment medical images to predict lesion-level treatment responses to chimeric antigen receptor (CAR) T-cell therapy in patients (or subjects) with lymphoma.
  • the lesion-level treatment response prediction is followed by a rule-based reasoning methodology for patient-level response prediction to CAR T-cell therapy.
  • Pre-treatment diagnostic CT images and PET/CT images of the neck, chest, abdomen, and pelvis previously obtained for clinical purposes in 39 (27M, 12F, 56.2 ⁇ 12.2 years) adult patients with lymphoma (with relapsed or refractory DLBCL or FL) who subsequently underwent CAR T-cell treatment with tisagenlecleucel (targeting CD 19+ cells) were utilized in this study.
  • This study included response prediction at the lesion-level and patient level. 26 patients (20M, 6F; median age 57 years (range 28-74))) with DFBCF and 13 patients (7M, 6F; median age 62 years (range43-72)) with FF were assessed for patient-level response prediction.
  • the patient inclusion and exclusion schema is shown in FIG. 1.
  • the identification of all individual lymph node disease sites was performed using pre treatment images followed by determination of ground truth lesion-level responses for all individual nodal lesions via comparison of pre-treatment and post-treatment images by an expert radiologist (DAT) with over 20 years of experience in the interpretation of CT, magnetic resonance imaging (MRI), and PET images.
  • DAT expert radiologist
  • Fesion-level response to treatment was defined by interval decrease in size or metabolic activity or interval resolution of a lesion between pretreatment and post-treatment images, whereas lesion-level non-response was defined as lack of change or interval increase in size or metabolic activity of a lesion.
  • the post-treatment dCT and PET/CT images utilized to determine ground truth lesion-level responses had been previously acquired 94.0 + 33.2 days after pre-treatment images.
  • IPI International Prognostic Index
  • FFIPI follicular lymphoma international prognostic index
  • Patients were categorized into three groups according to lesion responses detected on post- treatment CT or PET/CT images: (1) full responders (F-R), where all detected lesions demonstrated a response to treatment (i.e., either interval decrease in size, interval decrease in metabolic activity, or interval resolution); (2) full non-responders (F-NR), where all detected lesions demonstrated no response to treatment (i.e., either lack of change or interval increase in size or metabolic activity); and (3) partial responders (P-R), where some detected lesions demonstrated a response and others did not.
  • F-R full responders
  • F-NR full non-responders
  • P-R partial responders
  • the main study aim was to assess the feasibility of prediction of treatment response at the lesion level using a DL image analysis methodology, and following this, the feasibility of prediction of treatment response at the patient level using a rule-based reasoning approach.
  • dCT diagnostic computed tomography images
  • /Cl low-dose computed tomography images from PET/CT scans
  • PET positron emission tomography images from PET/CT scans.
  • VOI volume of interest
  • 770 402 by dCT + 214 by /CT+154 by PET lymph node lesions, as shown in Table 10, were assessed in this study.
  • 649 VOIs were placed around individual nodal lesions including 383 VOIs in pre-treatment dCT images, 158 VOIs in pre-treatment /CT images, and 108 VOIs in pre-treatment PET images.
  • VOI bounding box
  • the VOI operation was performed using CAVASS software [22] by setting up one rectangular box around the object/lymph node in 3D space. Every lesion was labeled with a 0 or 1 based on post-treatment response, where 0 indicated a lesion without response to treatment and 1 indicated a lesion with response to treatment.
  • a rectangular box was placed on the axial image slice with maximum cross-sectional area through a given abnormal lymph node, where the left-right and anterior-posterior margins were placed outside of the lymph node halfway from the lymph node center in the left-right and anterior-posterior directions, respectively.
  • the rectangular box was then propagated superiorly to one contiguous slice and inferiorly to one other contiguous slice.
  • lymph node lesions 402 by diagnostic computed tomography (dCT); 214 by low-dose CT (1CT); 154 by positron emission tomography (PET) were assessed (Table SI).
  • dCT diagnostic computed tomography
  • CT low-dose CT
  • PET positron emission tomography
  • 649 VOIs were placed around individual nodal lesions including 383 VOIs on dCT images, 158 VOIs on 1CT images, and 108 VOIs on PET images.
  • it was difficult to reliably place surrounding VOIs as the superior and inferior slices through the lymph nodes were unclear.
  • response predictions were performed using only whole-image slices as input.
  • transfer learning was employed for the prediction of treatment response. Transfer learning has been widely utilized in many DL applications, especially when there are limited training samples. Generally, transfer learning is performed by loading apre-trained neural network, followed by modifying its output layers/decision layers into layers for a specific classification purpose, and then retraining the whole network with specific training samples.
  • the flow chart of using transfer learning for this outcome prediction study is shown in FIG. 2. Although only the pre-trained neural network “AlexNet” [19] was used here, the same framework can be easily configured using other more recent pre-trained neural networks such as VGG16 [23] or ResNet [24]. AlexNet was selected for use in this study as it has a simpler structure (with only 5 convolutional layers) and is more easily retrained to test the proposed approach.
  • AlexNet was pre-trained on ImageNet data set in this study, which has 1 million images for image classification and 1000 image classes in total (involving natural and man-made objects).
  • AlexNet has 5 convolutional layers followed by rectified linear unit (ReLu) and Max Pooling layers, followed by 3 fully connected layers, another two ReLu and Dropout layers, and then a Soft-max layer before the final output layer for 1000 classes.
  • ReLu rectified linear unit
  • Max Pooling layers 3 fully connected layers
  • another two ReLu and Dropout layers and then a Soft-max layer before the final output layer for 1000 classes.
  • lesion-level treatment response was predicted as a binary classification as follows. The last three layers of the pre-trained network were previously configured for 1000 classes. All layers were extracted, except for the last three layers, from the pre-trained network.
  • the feature layers were transferred from the pre-trained network to the new classification task by replacing the last three layers with a fully connected layer, a Soft- max layer, and a binary classification output layer.
  • the options for the new fully connected layer such as number of epochs, batch size, etc., were specified according to the new training data.
  • AlexNet For AlexNet, different batch sizes of 5, 10, 20, and 30 were tested, with number of epochs of 40, 80, 100, and 200, using an initial learning rate for training as 10 ⁇ , and adopting a stochastic-gradient- descent- with-momentum algorithm for parameter optimization [25].
  • Image pre-processing operations included subtracting the mean and down-sampling using bilinear interpolation to 224 x 224 pixels from the original resolution.
  • the set of 383 VOI samples were divided from pre-treatment dCT data sets into training, validation, and testing data sets in the ratio of 6:2:2. Due to the limited training samples, a data augmentation technique [26] that is widely used and has been shown to be useful to improve training performance was adopted [27]. Data augmentation helped to prevent the network from overfitting and memorizing the exact details of the training images. All input images/slices were automatically resized into 224x224 size according to AlexNet input requirements. The data sets were then divided into training, validation, and testing data sets in the ratio of 6:2:2 (Table 11).
  • Augmentation operations on the training images included random flips of the training images along the vertical axis in the image slice (antero-posterior direction), random translations of up to 10 pixels horizontally and vertically (antero-posterior and right-to-left direction) within the slice plane, and affine operations scale change (magnification and minification) and rotation within the slice plane.
  • affine operations scale change magnification and minification
  • PET/C ZCT epochs [4080 100200] 0.6:0.2:0.2 Transfer learning with pre T Training: validation: trained AlexNet plus testing among 158 Incremental learning from
  • patient-level response analysis was performed by applying a rule -based reasoning approach on lesion-level prediction results from the DL network.
  • the predicted response for every lesion was first achieved by performing a leave-one- out (LOO) experiment at the lesion level. Then, after lesion-level response was predicted using transfer learning, two rules, the “All” rule and the “Majority” rule, were utilized to determine patient-level response as follows.
  • the “All” rule a patient responder is one in whom all lesions have responded, and a patient non-responder is one in whom at least one lesion has not responded.
  • the “Majority” rule a patient responder is one in whom the majority of lesions (using thresholds of either 60% or 70% of all lesions) have responded, and a patient non responder is one in whom the majority of lesions (using thresholds of either 60% or 70% of all lesions) have not responded.
  • the reference standard for patient-level response was based on the findings on cross-sectional imaging scans acquired up to 12-months after the date of pre treatment scans, which included patients with DLBCL (10 responder patients and 16 non responder patients) and patients with FL (8 responder patients and 5 non-responder patients). Since patient-level response assessment based on IPI (for patients with DLBCL) and FLIPI (for patients with FL) scores is currently used in clinical practice, we compared the performance of these clinical methods to our rule-based method in the 26 patients with DLBCL (10 responders and 16 non-responders).
  • IPI method DLBCL patients were categorized into responder and non- responder groups by using different score thresholds based on the number of IPI risk factors (of IPI ⁇ 1, IPI ⁇ 2, and IPI ⁇ 3), where the lower score groups were considered as the responder groups. The accuracy, sensitivity, and specificity of the patient-level prediction task were then evaluated for rule-based and IPI-based approaches, utilizing Pearson’s chi-square test for statistical comparisons.
  • FLIPI method patients were categorized into responder and non-responder groups by using different score thresholds (of FLIPI ⁇ 1, FLIPI ⁇ 2, FLIPI ⁇ 3, and FLIPI ⁇ 4), where the lower score groups were considered as the responder groups.
  • the transfer learning program was implemented in MATLAB 2018b and run under Ubuntu 16.04 OS on two computer systems including one PC with 64 GB RAM, 4 x Icore-7 CPUs, and two Nvidia 1080T ⁇ GPU cards with 22 GB GPU RAM in total, and another PC with 24 GB RAM, 4 x Icore-7 CPUs, and one Nvidia TITAN V GPU card with 12 GB GPU RAM.
  • the diagnostic performance results of lesion-level response prediction using transfer learning for the five scenarios from the dCt, /CT, and PET imaging modalities are shown in Table 12 (with p values provided in Table 13).
  • the predictive, diagnostic performances of the 1 VOI slice and 3 VOTslices input scenarios on dCt, /CT, and PET were substantially lower than those of the corresponding whole slice-based scenarios.
  • the accuracy of 1 VOTslice vs. 1 whole-slice input from dCT was 0.68+0.05 vs. 0.82+0.05, respectively (p ⁇ 0.0001) with AUC 0.59+0.04 vs. 0.91+0.03, respectively (p ⁇ 0.0001)
  • the accuracy of 3 VOTslices vs. 3 whole-slices input from dCT was 0.65+0.05 vs. 0.84+0.05, respectively (p ⁇ 0.0001) with AUC 0.52+0.07 vs. 0.90+0.05, respectively (p ⁇ 0.0001).
  • the predictive performances of 1 whole-slice and 3 whole-slices inputs from dCT (AUC 0.91+0.03 vs.
  • dCT diagnostic computed tomography
  • 1CT low-dose computed tomography
  • PET positron emission tomography
  • VOI volume of interest
  • Acc accuracy
  • Sens sensitivity
  • Spec specificity
  • AUC area under the curve.
  • the prediction performances using dCT, /CT, and PET were not statistically significantly different from each other.
  • the AUC of 1-slice from dCT, /CT, or PET was 0.91, 0.92 and 0.93, respectively, with p values of 0.71 (dCT vs. /CT), 0.42 (dCT vs. PET), and 0.79 (/CT vs. PET), and the AUC using 3-slices from dCT, /CT, or PET was 0.90, 0.94, and 0.95, respectively, with p values of 0.18 (dCT vs. /CT), 0.05 (dCT vs. PET) and 0.61 (/CT vs. PET). All p values are shown in Table 14. Table 14.
  • dCT diagnostic computed tomography
  • /CT low-dose computed tomography
  • PET positron emission tomography
  • Acc accuracy
  • Sens sensitivity
  • Spec specificity
  • AUC area under the curve.
  • ROC curves for the diagnostic performances of lesion-level treatment response prediction using transfer learning for four scenarios (1 VOI slice, 1 whole-slice, 3 VOI slices, and 3 whole-slices) using three different imaging modalities (dCT, /CT, and PET) are shown in FIG. 4.
  • ROC curves for the diagnostic performance of lesion-level treatment response prediction using transfer learning for the scenarios of 1 VOI slice plus 1 whole-slice and 1 whole-slice are shown in FIG. 5.
  • the diagnostic performances of 1 VOI slice plus 1 whole-slice using dCT, /CT, or PET were not statistically significantly different from those of 1 whole-slice (Table 15).
  • Details of the results of the five scenarios (1 VOI slice, 1 whole-slice, 3 VOI slices, 3 whole-slices, and 1 VOI slice plus 1 whole-slice (combined slices)) using three different imaging modalities are summarized in Table 16 (for dCT), Table 17 (for /CT), and Table 18 (for PET).
  • dCT diagnostic computed tomography
  • /CT low-dose computed tomography
  • PET positron emission tomography
  • Acc accuracy
  • Sens sensitivity
  • Spec specificity
  • AUC area under the curve
  • VOI volume of interest. Mean and standard deviation values were listed (mean ⁇ SD).
  • Table 17 Diagnostic performance of lesion-level treatment response prediction (using 40 epochs and batch size 5) based on input from 5 scenarios (1 VOI slice, 1 whole-slice, 3 VOI slices, 3 whole-slices, 1 VOI slice plus 1 whole-slice (combined slices)) from low-dose CT images of PET/CT.
  • VOI volume of interest
  • AUC area under the curve.
  • Table 18 Diagnostic performance of lesion-level treatment response prediction (using 40 epochs and batch size 5) based on input from 5 scenarios (1 VOI slice, 1 whole-slice, 3 VOI slices, 3 whole-slices, 1 VOI slice plus 1 whole-slice (combined slices)) from PET images of PET/CT.
  • VOI volume of interest
  • AUC area under the curve. Mean and standard deviation values were listed (mean ⁇ SD).
  • the diagnostic performance of lesion-level treatment response prediction based on input from 1 whole-slice dCT based on different parameters of batch size (B) and number of epochs (E) is shown in supplemental Table 20.
  • the program was run 10 times, and each time the whole data set was randomly separated into 60% training, 20% validation, and 20% testing, where the testing data sets were independent of the training and validation data sets.
  • Table 20 Diagnostic performance of lesion-level treatment response prediction using transfer learning on 1 whole-slice dCT based on different parameters of batch size (B) and number of epochs (E).
  • dCT diagnostic computed tomography
  • Acc accuracy
  • Sens sensitivity
  • Spec specificity
  • AUC area under the curve. Mean and standard deviation values were listed (mean ⁇ SD).

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Abstract

La présente invention concerne des procédés et des systèmes pour déterminer une réponse de traitement au niveau d'une lésion à un récepteur antigénique chimérique (CAR) par exemple, une thérapie CAR CD19, et les utilisations desdits procédés et systèmes pour évaluer la réactivité d'un sujet à une thérapie CAR CD19, et pour traiter un sujet avec une thérapie CAR CD19.
EP21714998.8A 2020-02-14 2021-02-12 Procédé de prédiction de réponse à une thérapie de récepteur antigénique chimérique Pending EP4104187A1 (fr)

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Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX374929B (es) 2013-02-20 2025-03-06 Novartis Ag RECEPTOR QUIMERICO DE ANTIGENO anti-EGFRvlll HUMANIZADO Y USOS DEL MISMO.
US9745368B2 (en) 2013-03-15 2017-08-29 The Trustees Of The University Of Pennsylvania Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
ES2963718T3 (es) 2014-01-21 2024-04-01 Novartis Ag Capacidad presentadora de antígenos de células CAR-T potenciada mediante introducción conjunta de moléculas co-estimuladoras
MY181834A (en) 2014-07-21 2021-01-08 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
AU2016245958B2 (en) 2015-04-08 2021-09-09 Novartis Ag CD20 therapies, CD22 therapies, and combination therapies with a CD19 Chimeric Antigen Receptor (CAR) - expressing cell
AU2016249005B2 (en) 2015-04-17 2022-06-16 Novartis Ag Methods for improving the efficacy and expansion of chimeric antigen receptor-expressing cells
US10829735B2 (en) 2015-07-21 2020-11-10 The Trustees Of The University Of Pennsylvania Methods for improving the efficacy and expansion of immune cells
BR112019006781A2 (pt) 2016-10-07 2019-07-30 Novartis Ag receptores de antígeno quiméricos para o tratamento de câncer
CA3079407A1 (fr) 2017-10-18 2019-04-25 Novartis Ag Compositions et methodes pour la degradation selective d'une proteine
CN112203725A (zh) 2018-06-13 2021-01-08 诺华股份有限公司 Bcma嵌合抗原受体及其用途
EP4065157A1 (fr) 2019-11-26 2022-10-05 Novartis AG Récepteurs antigéniques chimériques pour cd19 et cd22 et leurs utilisations
CN114675598B (zh) * 2022-03-29 2024-06-25 中南大学 基于迁移学习的不同数控机床刀尖模态参数预测方法及系统
CN119173527A (zh) * 2022-05-27 2024-12-20 凯德药业股份有限公司 细胞疗法构建体的非病毒递送

Family Cites Families (70)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2116183B (en) 1982-03-03 1985-06-05 Genentech Inc Human antithrombin iii dna sequences therefore expression vehicles and cloning vectors containing such sequences and cell cultures transformed thereby a process for expressing human antithrombin iii and pharmaceutical compositions comprising it
US6352694B1 (en) 1994-06-03 2002-03-05 Genetics Institute, Inc. Methods for inducing a population of T cells to proliferate using agents which recognize TCR/CD3 and ligands which stimulate an accessory molecule on the surface of the T cells
US6534055B1 (en) 1988-11-23 2003-03-18 Genetics Institute, Inc. Methods for selectively stimulating proliferation of T cells
US5858358A (en) 1992-04-07 1999-01-12 The United States Of America As Represented By The Secretary Of The Navy Methods for selectively stimulating proliferation of T cells
US6905680B2 (en) 1988-11-23 2005-06-14 Genetics Institute, Inc. Methods of treating HIV infected subjects
US5585362A (en) 1989-08-22 1996-12-17 The Regents Of The University Of Michigan Adenovirus vectors for gene therapy
DK0656946T4 (da) 1992-08-21 2010-07-26 Univ Bruxelles Immunoglobuliner uden lette kæder
US5350674A (en) 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
US7175843B2 (en) 1994-06-03 2007-02-13 Genetics Institute, Llc Methods for selectively stimulating proliferation of T cells
US5786464C1 (en) 1994-09-19 2012-04-24 Gen Hospital Corp Overexpression of mammalian and viral proteins
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
US6692964B1 (en) 1995-05-04 2004-02-17 The United States Of America As Represented By The Secretary Of The Navy Methods for transfecting T cells
US7067318B2 (en) 1995-06-07 2006-06-27 The Regents Of The University Of Michigan Methods for transfecting T cells
DE69739853D1 (de) 1996-02-28 2010-06-02 Ariad Pharma Inc Synthetische rapamycinderivate als multimerisierende wirkstoffe für chimere proteine mit von immunophilin abgeleiteten domänen
DE69738749D1 (de) 1996-08-16 2008-07-17 Schering Corp Zelloberflächen-antigen aus säugetieren und verwandte reagenzien
US6111090A (en) 1996-08-16 2000-08-29 Schering Corporation Mammalian cell surface antigens; related reagents
US6114148C1 (en) 1996-09-20 2012-05-01 Gen Hospital Corp High level expression of proteins
ES2301198T3 (es) 1997-06-12 2008-06-16 Novartis International Pharmaceutical Ltd. Polipeptidos artificiales de anticuerpos.
JP2001520039A (ja) 1997-10-21 2001-10-30 ヒューマン ジノーム サイエンシーズ， インコーポレイテッド ヒト腫瘍壊死因子レセプター様タンパク質、ｔｒ１１，ｔｒ１１ｓｖ１およびｔｒ１１ｓｖ２
IL137409A0 (en) 1998-02-09 2001-07-24 Genentech Inc Novel tumor necrosis factor receptor homolog and nucleic acids encoding the same
US20040040047A1 (en) 1998-03-30 2004-02-26 Spencer David M. Regulated apoptosis using chemically induced dimerization of apoptosis factors
DE60036945T2 (de) 1999-07-12 2008-08-21 Genentech, Inc., South San Francisco Stimulierung oder hemmung von angiogenese und herzvaskularisierung mit tumor nekrose faktor ligand/rezeptor homologen
AU1086501A (en) 1999-10-15 2001-04-30 Carnegie Institution Of Washington Rna interference pathway genes as tools for targeted genetic interference
US6326193B1 (en) 1999-11-05 2001-12-04 Cambria Biosciences, Llc Insect control agent
ES2302726T3 (es) 2000-02-24 2008-08-01 Invitrogen Corporation Estimulacion y concentracion simultanea de celulas.
US7572631B2 (en) 2000-02-24 2009-08-11 Invitrogen Corporation Activation and expansion of T cells
US6797514B2 (en) 2000-02-24 2004-09-28 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
US6867041B2 (en) 2000-02-24 2005-03-15 Xcyte Therapies, Inc. Simultaneous stimulation and concentration of cells
AU2001275474A1 (en) 2000-06-12 2001-12-24 Akkadix Corporation Materials and methods for the control of nematodes
CN1294148C (zh) 2001-04-11 2007-01-10 中国科学院遗传与发育生物学研究所 环状单链三特异抗体
CA2763913C (fr) 2001-08-10 2014-10-28 Aberdeen University Domaines de liaison a l'antigene
US7745140B2 (en) 2002-01-03 2010-06-29 The Trustees Of The University Of Pennsylvania Activation and expansion of T-cells using an engineered multivalent signaling platform as a research tool
US7446190B2 (en) 2002-05-28 2008-11-04 Sloan-Kettering Institute For Cancer Research Nucleic acids encoding chimeric T cell receptors
US7618632B2 (en) 2003-05-23 2009-11-17 Wyeth Method of treating or ameliorating an immune cell associated pathology using GITR ligand antibodies
EP1638510B1 (fr) 2003-07-01 2015-09-02 Immunomedics, Inc. Porteuses polyvalentes d'anticorps bispecifiques
US20050048054A1 (en) 2003-07-11 2005-03-03 Shino Hanabuchi Lymphocytes; methods
US7435596B2 (en) 2004-11-04 2008-10-14 St. Jude Children's Research Hospital, Inc. Modified cell line and method for expansion of NK cell
EP1692318A4 (fr) 2003-12-02 2008-04-02 Genzyme Corp Compositions et methodes pour le diagnostic et le traitement du cancer du poumon
GB0409799D0 (en) 2004-04-30 2004-06-09 Isis Innovation Method of generating improved immune response
EP2295588B1 (fr) 2004-05-27 2018-03-07 The Trustees Of The University Of Pennsylvania Nouvel antigène artificiel présentant des cellules et utilisations associées
US20060002932A1 (en) 2004-06-04 2006-01-05 Duke University Methods and compositions for enhancement of immunity by in vivo depletion of immunosuppressive cell activity
EP1786918A4 (fr) 2004-07-17 2009-02-11 Imclone Systems Inc Nouveau anticorps bispecifique tetravalent
EP1866339B8 (fr) 2005-03-25 2021-12-01 GITR, Inc. Molecules de liaison gitr et leurs utilisations
EP2520588A1 (fr) 2005-08-19 2012-11-07 Abbott Laboratories Immunoglobuline à double domaine variable et ses utilisations
EP1981969A4 (fr) 2006-01-19 2009-06-03 Genzyme Corp Anticorps anti-gitr destines au traitement du cancer
KR100806107B1 (ko) 2006-04-11 2008-02-21 엘지전자 주식회사 Ｄｒｍ에서의 비보호 콘텐츠의 보호 방법
EP3124046B1 (fr) 2007-07-12 2019-12-25 GITR, Inc. Thérapies combinées utilisant des molécules de liaison gitr
MX2011000039A (es) 2008-07-02 2011-05-31 Emergent Product Dev Seattle Proteinas antagonistas del factor-beta de crecimiento transformante (tgf-beta), que se unen a multiples objetivos.
CN102149820B (zh) 2008-09-12 2014-07-23 国立大学法人三重大学 能够表达外源gitr配体的细胞
AU2010289677B2 (en) 2009-09-03 2014-07-31 Merck Sharp & Dohme Llc Anti-GITR antibodies
GB0919054D0 (en) 2009-10-30 2009-12-16 Isis Innovation Treatment of obesity
TR201904484T4 (tr) 2009-11-03 2019-05-21 Hope City Transdüse T hücre seçimine yönelik kesik epiderimal büyüme faktörü reseptörü (EGFRt).
CN105693861A (zh) 2009-12-29 2016-06-22 新兴产品开发西雅图有限公司 异二聚体结合蛋白及其应用
US9089520B2 (en) 2010-05-21 2015-07-28 Baylor College Of Medicine Methods for inducing selective apoptosis
AU2012240562B2 (en) 2011-04-08 2016-12-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Anti-epidermal growth factor receptor variant III chimeric antigen receptors and use of same for the treatment of cancer
US20130108641A1 (en) 2011-09-14 2013-05-02 Sanofi Anti-gitr antibodies
TW201414837A (zh) 2012-10-01 2014-04-16 Univ Pennsylvania 標定基質細胞以治療癌症之組合物和方法
US10117896B2 (en) 2012-10-05 2018-11-06 The Trustees Of The University Of Pennsylvania Use of a trans-signaling approach in chimeric antigen receptors
LT2956175T (lt) 2013-02-15 2017-12-11 The Regents Of The University Of California Chimerinis antigeno receptorius ir jo panaudojimo būdai
US9434935B2 (en) 2013-03-10 2016-09-06 Bellicum Pharmaceuticals, Inc. Modified caspase polypeptides and uses thereof
CA2905352A1 (fr) 2013-03-14 2014-09-25 Bellicum Pharmaceuticals, Inc. Procedes de regulation de la proliferation cellulaire
US9745368B2 (en) 2013-03-15 2017-08-29 The Trustees Of The University Of Pennsylvania Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
TWI654206B (zh) 2013-03-16 2019-03-21 諾華公司 使用人類化抗－ｃｄ１９嵌合抗原受體治療癌症
US9701758B2 (en) 2013-05-24 2017-07-11 Board Of Regents, The University Of Texas System Anti-CD19 scFv (FMC63) polypeptide
EP3004329B1 (fr) 2013-06-05 2020-03-04 Bellicum Pharmaceuticals, Inc. Procédé d'induction d'une apoptose partielle au moyen de polypeptides de caspase
JP6793902B2 (ja) 2013-12-20 2020-12-02 ノバルティス アーゲー 調節可能キメラ抗原受容体
WO2015142675A2 (fr) 2014-03-15 2015-09-24 Novartis Ag Traitement du cancer au moyen d'un récepteur antigénique chimérique
US10282588B2 (en) * 2016-06-09 2019-05-07 Siemens Healthcare Gmbh Image-based tumor phenotyping with machine learning from synthetic data
WO2019104217A1 (fr) * 2017-11-22 2019-05-31 The Trustees Of Columbia University In The City Of New York Procédé de système et support accessible par ordinateur pour classifier un tissu mammaire au moyen d'un réseau neuronal convolutionnel
WO2020028382A1 (fr) * 2018-07-30 2020-02-06 Memorial Sloan Kettering Cancer Center Réseaux neuronaux d'apprentissage profond multimode, multi-résolution pour la segmentation, la prédiction de résultats, et la surveillance de réponses longitudinales à l'immunothérapie et à la radiothérapie

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